Eduardo Lapetina - Academia.edu (original) (raw)
Papers by Eduardo Lapetina
Advances in Experimental Medicine and Biology, 1976
Enhanced labelling of phosphatidylinositol occurs in different type of cells during stiinulation ... more Enhanced labelling of phosphatidylinositol occurs in different type of cells during stiinulation (1,2). This effect includes phosphatidylinositol breakdown and resynthesis (see cycle of reaction in Hawthorne et al., this Synposium) and has recently been related in various tissues to α -adrenergic and muscarinic cholinergic agonists (2). According to the available information it seems most probably that the site of control of this effect is confined to phosphatidylinositol breakdown to 1,2-diacylglycerol. Pancreas, parotid and longitudinal ileum smooth muscle show a fall in the concentration of phosphatidylinositol during stimulation (3,4,5). It is then possible that studies of phosphatidylinositol labelling are only a reflection of the primary event which is phosphatidylinositol breakdown. A specific phospholipase-C type of activity against phosphatidylinositol has been found in particulate fractions of brain and vas deferens smooth muscle (6-8). This activity produces breakdown of phosphatidylinositol with production of 1,2- diacylglycerol, myoinositol 1,2-cyclic phosphate and myoinositol 1-phosphate (8,9)
Cellular Signalling, Feb 1, 1991
The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects... more The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.
Biochem Biophys Res Commun, 1985
The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmito... more The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.
Proceedings of the National Academy of Sciences of the United States of America, 1986
The Journal of Immunology, Jul 15, 1999
Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in th... more Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2 ؉ , and Bax ؉ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-␣ and nitric oxide. TUNEL ؉ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68 ؉ and myeloperoxidase ؉ , but no CD45RO ؉ , apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivationinduced apoptosis. CD T cells were also more resistant to Fas-and nitric oxide-mediated apoptosis, whereas TNF-␣ failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2 ؉ , but fewer Bax ؉ , cells, while UC mucosa contained fewer Bcl-2 ؉ , but more Bax ؉ , cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.
Endocrinology, Apr 1, 1992
Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell lin... more Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell line that overexpresses human IGF-I receptors, led to an increase in the amount of a phosphatidylinositol kinase that could be immunoprecipitated by anti-IGF-I receptor or anti-phosphotyrosine antibodies. The identity of the lipid produced in phosphatidylinositol kinase assays of anti-IGF-I receptor or anti-phosphotyrosine immunoprecipitates indicated that IGF-I selectively increased the amount of immunoprecipitated phosphatidylinositol 3-kinase activity. The amount of immunoprecipitated phosphatidylinositol 3-kinase activity that was increased by IGF-I followed a time course that paralleled the stimulation of IGF-I receptor beta-subunit autophosphorylation. The amount of phosphatidylinositol 3-kinase activity detected in anti-IGF-I receptor immunoprecipitates represented only 2% of that which was immunoprecipitated by anti-phosphotyrosine antibody. Furthermore, phosphatidylinositol 3-kinase activity which was recovered with anti-phosphotyrosine antibody was present in both cytosol and particulate cell fractions at approximately similar levels. Taken together, these results suggest that the stimulation of the IGF-I receptor tyrosine kinase leads to an increase in the amount of phosphatidyl inositol 3-kinase activity immunoprecipitated by antiphosphotyrosine and anti-IGF-I receptor antibodies and to a limited association with the IGF-I receptor itself, even though these cells express very high levels of IGF-I receptors. That the majority of phosphatidylinositol 3-kinase activity does not tightly associate with the IGF-I receptor after IGF-I stimulation suggests that it may be associated with other tyrosine phosphorylated proteins. Alternatively, the kinase itself may become phosphorylated on tyrosine and dissociate from the IGF-I receptor. In this manner, an increase of phosphatidylinositol 3-kinase activity by IGF-I deviates from the activation of phosphatidylinositol 3-kinase by platelet-derived growth factor receptor in that a tight association with the receptor is not produced after stimulation.
Biochimica Et Biophysica Acta General Subjects, Nov 1, 1986
It has recently been shown in this laboratory that permeabilization of human platelets with 15-25... more It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 #g/ml saponin allows ADP-ribosylation by pertussis toxin of the ai-subunit of G i (N 0, a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate-(GTPIySI-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3Hlinositol. Phospholipase C activity was measured by [3Hlpolyphosphoinositide decreases and formation of [3Hlinositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the ai-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Grprotein was not directly related to activation or inhibition of platelet phospholipase C by GTPITS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.
Annals of the New York Academy of Sciences, May 1, 1994
Page 1. Platelet Activation and Inhibition Novel Signal Transduction Mechanisms SCOTT N. PETERSON... more Page 1. Platelet Activation and Inhibition Novel Signal Transduction Mechanisms SCOTT N. PETERSON AND EDUARDO G. LAPETINA Division of Cell Biology Burroughs Wellcome Co. ... 1989. Cell 57: 30. FARRELL, F. X., M. TORTI & EG LAPETINA. 1992. J. Lab. Clin. Med. ...
Biochemical Journal, Dec 1, 1985
Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into itscorresponding phosphatidic acid and ... more Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into itscorresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. l-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3,M upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 /SM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 /M-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.
Biochemical and Biophysical Research Communications, Dec 16, 1991
Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained ... more Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.
Proceedings of the National Academy of Sciences of the United States of America, Apr 28, 1998
Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell... more Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donor S-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.
Biochemical and Biophysical Research Communications, Oct 29, 1987
Biochemical and Biophysical Research Communications, 1990
The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,&bisphosphate i... more The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,&bisphosphate in washed human platelets was studied. Platelets labelled with [32~]~i were stimulated with phorbol 12,13-dibutyrate or thrombin in the presence or absence of staurosporine. Lipids were extracted, and deacylated, and the glycerophosphoinositol derivatives were analyzed by high performance liquid chromatography. Phorbol 12,13-dibutyrate increased formation of phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4-bisphosphate in a dose-and time-dependent manner. Thrombin also increased formation of phosphatidylinositol 3,4-bisphosphate. Staurosporine completely inhibited phorbol 12,13-dibutyrate or thrombinstimulated production of phosphatidylinositol 3,4-bisphosphate. These data indicate that production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4-monophosphate is mediated by protein kinase C. It is widely recognized that production of phosphatidylinositol 3,4-bisphosphate is caused by the tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase. However,in platelets, production of phosphatidylinositol 3,4-bisphosphate might be related to stimulation of phosphatidylinositol 4-kinase, which is activated by protein kinase C. '01990 Academic Press, Inc. Recent studies have shown the presence of phosphatidylinositol (PI) 3-kinase in various cell types, This enzyme phosphorylates at the D-3 position on the inositol ring(l), has an important role in cell transformation(2), and has been identified because of its specific and tight association with certain activated protein-tyrosine kinases, including pp6Gv-src, polyoma virus middle T/pp60c-src, platelet derived growth
Biochimica Et Biophysica Acta, 1986
Phosphatidic acid (PA) is an obligate intermediate in the phosphatidylinositol (PI) cycle and may... more Phosphatidic acid (PA) is an obligate intermediate in the phosphatidylinositol (PI) cycle and may serve as a sensitive marker for this pathway of reactions. Previously, we have shown that acetylcholine (ACh) stimulates formation of labeled PA in whole rat corneas whose phospholipids were prelabeled with [14C]arachidonate. To determine which layer of the cornea exhibits the ACh effect, intact epithelium was isolated from the rest of the corneal tissue (designated "stroma/endothelium") by incubating rat corneas with neutral protease and then stripping off the epithelium using forceps. The epithelium was ultrastructurally normal and avidly incorporated [14C]arachidonate into phospholipids; the stroma/endothelium had only trace incorporation. [14C]Arachidonyl-PA formation by the epithelium was significantly increased after a 37 second (+58%) and was maximal after a 5.0 minute (+188%) incubation in the presence of 1 mM ACh. The stimulation by ACh was blocked by atropine and scopolamine but not by d-tubocurarine. The epithelium also incorporated significant quantities of [3H]inositol into phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and phosphatidyl-inositol bisphosphate (PIP 2). When [3H]inositol-labeled epithelia were incubated for 5 minutes with 1 mM ACh, [3H]inositol monophosphate (IP) was increased 200%, [3H]inositol bisphosphate (IP2) was increased 225%, and [3H]inositol 2 trisphosphate (IP3) was increased 74%. These results suggest that muscarinic cholinergic receptors may be an important regulator of the PI cycle in corneal epithelium and thus may affect intracellular calcium mobilization and epithelial proliferation and regeneration.
Biochemical and Biophysical Research Communications, Jun 18, 1998
Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the mac... more Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the macrophage cell line RAW 267.4. RES cells that have been derived from RAW 267.4 cells by repeated exposure to lipopolysaccharide and interferon-gamma (LPS/INF-gamma), followed by outgrowth of viable cells, are resistant to apoptosis and caspase-3 activation upon exposure to SpNO. In this study we have determined that RES cells have lower levels of glutathione (GSH) and a higher oxidative state than RAW cells. Subsequently, RAW and RES cells were depleted of GSH by using l-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. GSH depleted cells did not undergo apoptosis nor demonstrate caspase-3 activity when they were exposed to SpNO. These results suggest that the redox status of the cell is one of the key factors mediating the apoptotic pathway in which glutathione plays a critical role in mediating apoptosis via NO* and reactive oxygen species (ROS).
Ann N Y Acad Sci, 1989
Arachidonic acid can be released from membrane phospholipids of platelets in response to a number... more Arachidonic acid can be released from membrane phospholipids of platelets in response to a number of receptor-mediated signals. The enzymes most responsible for this activation are phospholipase A2' and, to a lesser degree, 1,2-diacylgIycerol lipase.' In this paper, we will describe some of the studies on phospholipase A2 activation that we have carried out and also some other published work that has helped us understand some of the physiological control mechanisms of this enzyme. The importance of phospholipase A, in receptor-mediated platelet activation varies with both the type and the strength of agonist used. As examples, collagen and epinephrine are absolutely dependent on the release of arachidonic acid for stimulation of platelet secretion and aggregation, whereas thrombin depends on arachidonic acid release only when used at low concentration; at higher doses the ability of thrombin to activate platelets is independent of arachidonic acid metabolites. The most relevant arachidonic acid metabolites for platelet stimulation are endoperoxides and thromboxane A'.
Biochem Biophys Res Commun, 1987
The American journal of physiology, 1998
Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epi... more Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachm...
Advances in Experimental Medicine and Biology, 1976
Enhanced labelling of phosphatidylinositol occurs in different type of cells during stiinulation ... more Enhanced labelling of phosphatidylinositol occurs in different type of cells during stiinulation (1,2). This effect includes phosphatidylinositol breakdown and resynthesis (see cycle of reaction in Hawthorne et al., this Synposium) and has recently been related in various tissues to α -adrenergic and muscarinic cholinergic agonists (2). According to the available information it seems most probably that the site of control of this effect is confined to phosphatidylinositol breakdown to 1,2-diacylglycerol. Pancreas, parotid and longitudinal ileum smooth muscle show a fall in the concentration of phosphatidylinositol during stimulation (3,4,5). It is then possible that studies of phosphatidylinositol labelling are only a reflection of the primary event which is phosphatidylinositol breakdown. A specific phospholipase-C type of activity against phosphatidylinositol has been found in particulate fractions of brain and vas deferens smooth muscle (6-8). This activity produces breakdown of phosphatidylinositol with production of 1,2- diacylglycerol, myoinositol 1,2-cyclic phosphate and myoinositol 1-phosphate (8,9)
Cellular Signalling, Feb 1, 1991
The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects... more The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.
Biochem Biophys Res Commun, 1985
The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmito... more The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.
Proceedings of the National Academy of Sciences of the United States of America, 1986
The Journal of Immunology, Jul 15, 1999
Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in th... more Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2 ؉ , and Bax ؉ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-␣ and nitric oxide. TUNEL ؉ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68 ؉ and myeloperoxidase ؉ , but no CD45RO ؉ , apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivationinduced apoptosis. CD T cells were also more resistant to Fas-and nitric oxide-mediated apoptosis, whereas TNF-␣ failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2 ؉ , but fewer Bax ؉ , cells, while UC mucosa contained fewer Bcl-2 ؉ , but more Bax ؉ , cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.
Endocrinology, Apr 1, 1992
Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell lin... more Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell line that overexpresses human IGF-I receptors, led to an increase in the amount of a phosphatidylinositol kinase that could be immunoprecipitated by anti-IGF-I receptor or anti-phosphotyrosine antibodies. The identity of the lipid produced in phosphatidylinositol kinase assays of anti-IGF-I receptor or anti-phosphotyrosine immunoprecipitates indicated that IGF-I selectively increased the amount of immunoprecipitated phosphatidylinositol 3-kinase activity. The amount of immunoprecipitated phosphatidylinositol 3-kinase activity that was increased by IGF-I followed a time course that paralleled the stimulation of IGF-I receptor beta-subunit autophosphorylation. The amount of phosphatidylinositol 3-kinase activity detected in anti-IGF-I receptor immunoprecipitates represented only 2% of that which was immunoprecipitated by anti-phosphotyrosine antibody. Furthermore, phosphatidylinositol 3-kinase activity which was recovered with anti-phosphotyrosine antibody was present in both cytosol and particulate cell fractions at approximately similar levels. Taken together, these results suggest that the stimulation of the IGF-I receptor tyrosine kinase leads to an increase in the amount of phosphatidyl inositol 3-kinase activity immunoprecipitated by antiphosphotyrosine and anti-IGF-I receptor antibodies and to a limited association with the IGF-I receptor itself, even though these cells express very high levels of IGF-I receptors. That the majority of phosphatidylinositol 3-kinase activity does not tightly associate with the IGF-I receptor after IGF-I stimulation suggests that it may be associated with other tyrosine phosphorylated proteins. Alternatively, the kinase itself may become phosphorylated on tyrosine and dissociate from the IGF-I receptor. In this manner, an increase of phosphatidylinositol 3-kinase activity by IGF-I deviates from the activation of phosphatidylinositol 3-kinase by platelet-derived growth factor receptor in that a tight association with the receptor is not produced after stimulation.
Biochimica Et Biophysica Acta General Subjects, Nov 1, 1986
It has recently been shown in this laboratory that permeabilization of human platelets with 15-25... more It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 #g/ml saponin allows ADP-ribosylation by pertussis toxin of the ai-subunit of G i (N 0, a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate-(GTPIySI-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3Hlinositol. Phospholipase C activity was measured by [3Hlpolyphosphoinositide decreases and formation of [3Hlinositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the ai-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Grprotein was not directly related to activation or inhibition of platelet phospholipase C by GTPITS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.
Annals of the New York Academy of Sciences, May 1, 1994
Page 1. Platelet Activation and Inhibition Novel Signal Transduction Mechanisms SCOTT N. PETERSON... more Page 1. Platelet Activation and Inhibition Novel Signal Transduction Mechanisms SCOTT N. PETERSON AND EDUARDO G. LAPETINA Division of Cell Biology Burroughs Wellcome Co. ... 1989. Cell 57: 30. FARRELL, F. X., M. TORTI & EG LAPETINA. 1992. J. Lab. Clin. Med. ...
Biochemical Journal, Dec 1, 1985
Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into itscorresponding phosphatidic acid and ... more Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into itscorresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. l-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3,M upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 /SM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 /M-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.
Biochemical and Biophysical Research Communications, Dec 16, 1991
Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained ... more Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.
Proceedings of the National Academy of Sciences of the United States of America, Apr 28, 1998
Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell... more Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donor S-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.
Biochemical and Biophysical Research Communications, Oct 29, 1987
Biochemical and Biophysical Research Communications, 1990
The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,&bisphosphate i... more The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,&bisphosphate in washed human platelets was studied. Platelets labelled with [32~]~i were stimulated with phorbol 12,13-dibutyrate or thrombin in the presence or absence of staurosporine. Lipids were extracted, and deacylated, and the glycerophosphoinositol derivatives were analyzed by high performance liquid chromatography. Phorbol 12,13-dibutyrate increased formation of phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4-bisphosphate in a dose-and time-dependent manner. Thrombin also increased formation of phosphatidylinositol 3,4-bisphosphate. Staurosporine completely inhibited phorbol 12,13-dibutyrate or thrombinstimulated production of phosphatidylinositol 3,4-bisphosphate. These data indicate that production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4-monophosphate is mediated by protein kinase C. It is widely recognized that production of phosphatidylinositol 3,4-bisphosphate is caused by the tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase. However,in platelets, production of phosphatidylinositol 3,4-bisphosphate might be related to stimulation of phosphatidylinositol 4-kinase, which is activated by protein kinase C. '01990 Academic Press, Inc. Recent studies have shown the presence of phosphatidylinositol (PI) 3-kinase in various cell types, This enzyme phosphorylates at the D-3 position on the inositol ring(l), has an important role in cell transformation(2), and has been identified because of its specific and tight association with certain activated protein-tyrosine kinases, including pp6Gv-src, polyoma virus middle T/pp60c-src, platelet derived growth
Biochimica Et Biophysica Acta, 1986
Phosphatidic acid (PA) is an obligate intermediate in the phosphatidylinositol (PI) cycle and may... more Phosphatidic acid (PA) is an obligate intermediate in the phosphatidylinositol (PI) cycle and may serve as a sensitive marker for this pathway of reactions. Previously, we have shown that acetylcholine (ACh) stimulates formation of labeled PA in whole rat corneas whose phospholipids were prelabeled with [14C]arachidonate. To determine which layer of the cornea exhibits the ACh effect, intact epithelium was isolated from the rest of the corneal tissue (designated "stroma/endothelium") by incubating rat corneas with neutral protease and then stripping off the epithelium using forceps. The epithelium was ultrastructurally normal and avidly incorporated [14C]arachidonate into phospholipids; the stroma/endothelium had only trace incorporation. [14C]Arachidonyl-PA formation by the epithelium was significantly increased after a 37 second (+58%) and was maximal after a 5.0 minute (+188%) incubation in the presence of 1 mM ACh. The stimulation by ACh was blocked by atropine and scopolamine but not by d-tubocurarine. The epithelium also incorporated significant quantities of [3H]inositol into phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and phosphatidyl-inositol bisphosphate (PIP 2). When [3H]inositol-labeled epithelia were incubated for 5 minutes with 1 mM ACh, [3H]inositol monophosphate (IP) was increased 200%, [3H]inositol bisphosphate (IP2) was increased 225%, and [3H]inositol 2 trisphosphate (IP3) was increased 74%. These results suggest that muscarinic cholinergic receptors may be an important regulator of the PI cycle in corneal epithelium and thus may affect intracellular calcium mobilization and epithelial proliferation and regeneration.
Biochemical and Biophysical Research Communications, Jun 18, 1998
Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the mac... more Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the macrophage cell line RAW 267.4. RES cells that have been derived from RAW 267.4 cells by repeated exposure to lipopolysaccharide and interferon-gamma (LPS/INF-gamma), followed by outgrowth of viable cells, are resistant to apoptosis and caspase-3 activation upon exposure to SpNO. In this study we have determined that RES cells have lower levels of glutathione (GSH) and a higher oxidative state than RAW cells. Subsequently, RAW and RES cells were depleted of GSH by using l-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. GSH depleted cells did not undergo apoptosis nor demonstrate caspase-3 activity when they were exposed to SpNO. These results suggest that the redox status of the cell is one of the key factors mediating the apoptotic pathway in which glutathione plays a critical role in mediating apoptosis via NO* and reactive oxygen species (ROS).
Ann N Y Acad Sci, 1989
Arachidonic acid can be released from membrane phospholipids of platelets in response to a number... more Arachidonic acid can be released from membrane phospholipids of platelets in response to a number of receptor-mediated signals. The enzymes most responsible for this activation are phospholipase A2' and, to a lesser degree, 1,2-diacylgIycerol lipase.' In this paper, we will describe some of the studies on phospholipase A2 activation that we have carried out and also some other published work that has helped us understand some of the physiological control mechanisms of this enzyme. The importance of phospholipase A, in receptor-mediated platelet activation varies with both the type and the strength of agonist used. As examples, collagen and epinephrine are absolutely dependent on the release of arachidonic acid for stimulation of platelet secretion and aggregation, whereas thrombin depends on arachidonic acid release only when used at low concentration; at higher doses the ability of thrombin to activate platelets is independent of arachidonic acid metabolites. The most relevant arachidonic acid metabolites for platelet stimulation are endoperoxides and thromboxane A'.
Biochem Biophys Res Commun, 1987
The American journal of physiology, 1998
Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epi... more Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachm...