Elizabeth Kornecki

Long-term potentiation in the hippocampus induced by platelet-activating factor

Platelet-activating factor (PAF) is an ether phospholipid that serves as an autacoid in a wide range of biological processes. We found that when PAF was added to hippocampal slices, it induced a stable and concentration-dependent increase in excitatory postsynaptic potential and population spike recordings (long-term potentiation [LTP]). The PAF effect was blocked by the PAF receptor antagonists BN 52021 and WEB 2086 and the N-methyl-D-aspartate receptor antagonists MK 801 and 2-amino-5-phosphonovalerate. However, these PAF receptor antagonists did not block LTP induced by high frequency stimulation. The facilitation induced by PAF could not be reversed by PAF receptor antagonists. Induction of either PAF-or tetanus-triggered facilitation occluded the subsequent expression of the other, suggesting a common pathway. LTP is a type of synaptic plasticity associated with the formation of memory, and PAF may play an important modulatory role in this process.

Ectoprotein kinase in the regulation of cellular responsiveness to extracellular ATP.

The reversible phosphorylation of intracellular proteins has been established as a key regulatory mechanism in numerous cellular functions. In this process the enzyme protein kinase transfers the gamma-phosphate of ATP to form a covalent bond with specific proteins. Another line of investigation has demonstrated that extracellular ATP is a potent physiological regulator in various cellular systems. Although many of the physiological effects of extracellular ATP were shown to be mediated by the action of purinergic receptors, it is possible that extracellular protein phosphorylation systems are also implicated in the mechanisms underlying the responsiveness of cells to extracellular ATP. The identification of ectoprotein kinase at the surface of various cells has provided evidence for the existence of such mechanisms, and revealed how the regulatory powers of protein phosphorylation systems can extend to the extracellular environment. The versatile roles that extracellular protein phosphorylation activity may play in the regulation of cellular functions is underscored by the presence of multiple protein substrates for this activity at the cell surface. Each such surface phosphoprotein may have a unique function. FIGURE 5 depicts the hypothetical relationships between the extracellular ATP secreted by exocytosis and the specific physiological function of these secreting neurons. Based on findings described in this article, we propose that extracellular ATP can be utilized by two types of extracellular protein kinase: a membrane-bound ectoprotein kinase, and a soluble exoprotein kinase. The exoprotein kinase can originate by detachment of an ectokinase from the cell surface, or be an intravesicular protein that is coreleased with ATP by exocytosis from stimulated cells. Phosphorylation of specific proteins at the surface of a secreting cell may have an important feedback control over its own presynaptic activity. The ectoprotein kinase could exert this feedback regulation by phosphorylating ion channels involved in secretion, and/or by phosphorylating transporters that carry out the reuptake of released transmitter molecules. Phosphorylation of receptors can regulate intercellular communication, and phosphorylation of integrins could regulate the interaction of the cell surface with components of the extracellular matrix. Although most of the relationships suggested in FIGURE 5 are still hypothetical, it should be possible to test them experimentally in a direct manner by raising antibodies against the phosphorylated sites of specific surface phosphoproteins. The ability of such antibodies to inhibit protein phosphorylation without penetrating the cells provides an experimental paradigm for the direct testing of potential physiological function of ecto- and exoprotein kinase activities in a variety of cells.

Surface Phosphorylation by Ecto-Protein Kinase C in Brain Neurons: A Target for Alzheimer's β-Amyloid Peptides

Abstract: The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ectoprotein kinase (ecto‐PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC‐pseudosubstrate 19–36; K252b) and antibodies (anti‐PKC catalytic region M.Ab.1.9, antibodies to the carboxy‐terminus of eight PKC isozymes) revealed several types of ecto‐PK activity, among them ecto‐PKs with catalytic specificity of the PKC isozymes ζ and δ. The activity of the neuronal ecto‐PKC is constitutive and not stimulated by phorbol esters. The phosphorylation of a 12K/13K surface protein duplex by ecto‐PKC‐δ was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimers amyloid peptides β1–40 and β25–35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto‐PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose‐response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, β‐amyloid peptide 1–28 did not inhibit this surface phosphorylation. The discovery that ecto‐PKC‐mediated protein phosphorylation serves as a target for β‐amyloid peptides at the very site they operate, i.e., at the neuronal cell surface, opens a new research direction in the investigation of molecular events that play a role in the etiology of developmental disabilities and neurodegenerative disorders.

Signaling Pathways of the F11 Receptor (F11R; a.k.a. JAM-1, JAM-A) in Human Platelets: F11R Dimerization, Phosphorylation and Complex Formation with the Integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the FcγRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the FcγRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the FcγRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.

The F11 receptor (F11R/JAM-A) in atherothrombosis: Overexpression of F11R in atherosclerotic plaques

F11R is the gene name for an adhesion protein, called the F11-receptor, aka JAM-A, which under normal physiological conditions is expressed constitutively on the surface of platelets and localized within tight junctions of endothelial cells (EC). Previous studies of the interactions between human platelets and EC suggested that F11R/JAM-A plays a crucial role in inflammatory thrombosis and atherosclerosis. The study reported here obtained in-vivo confirmation of this conclusion by investigating F11R/JAM-A protein and mRNA in patients with aortic and peripheral vascular disease and in an animal model of atherosclerosis. Molecular and immunofluorescence determinations revealed very high levels of F11R/JAM-A mRNA and F11R/JAM-A protein in atherosclerotic plaques of cardiovascular patients. Similar results were obtained with 12-week-old atherosclerosis-prone apoE-/- mice, an age in which atherosclerotic plaques are well established. Enhanced expression of the F11R/JAM-A message in cultured EC from human aortic and venous vessels was observed following exposure of the cells to cytokines. Determinations of platelet adhesion to cultured EC inflamed by combined cytokine treatment in the presence of F11R/JAM-A - antagonists provided data indicating that de novo expression of F11R/JAM-A on the luminal surface of inflamed EC has an important role in the conversion of EC to a thrombogenic surface. Further studies of these interactions under flow conditions and under in-vivo settings could provide a final proof of a causal role for F11R/JAM-A in the initiation of thrombosis. Based on our in-vitro and in-vivo studies to date, we propose that therapeutic drugs which antagonize the function of F11R/JAM-A should be tested as novel means for the prevention and treatment of atherosclerosis, heart attacks and stroke.

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrodes buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.

Calcium ion mobilization in neuronal cells induced by PAF

We have reported previously that platelet-activating factor (PAF) interacts with the neuronal cell line NG108-15 (neuroblastoma X glioma hybrid) and the pheochromocytoma cell line, PC12. PAF acts on these cells by raising levels of intracellular free calcium ions. In the present report, we extend these studies. PAF induced the vesicular release of adenosine 5′-triphosphate (ATP) from PC12 cells in a dose-dependent manner. The PAF-induced ATP release was inhibited by the PAF antagonists, CV-3988 and CV-6209, and the calcium antagonist prenylamine. The relevance of the interaction of PAF with neuronal cells was investigated further by using brain synaptosomal preparations and primary cortical and neostriatal cells. Nanomolar concentrations of PAF induced calcium transients in aequorin-loaded synaptosomal preparations, and cortical and neostriatal cells were sensitive to the action of PAF. The possible physiological and pathophysiological roles of PAF in brain function are discussed.

Ecto-protein kinase and surface protein phosphorylation in PC12 cells: interactions with nerve growth factor.

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ‐32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto‐protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho‐α‐casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto‐protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto‐protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+‐dependent, high‐affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K‐252a inhibited both intra‐ and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K‐252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto‐protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto‐protein kinase in the reported inhibition of NGF‐induced neurite extension by K‐252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto‐protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto‐protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF‐induced neuronal differentiation. In the concentration range (10–100 μM) at which 6‐thioguanine blocked NGF‐promoted neurite outgrowth in PC12 cells, 6‐thioguanine effectively inhibited the phosphorylation of specific proteins by ecto‐protein kinase. This study provides the basis for continued investigation of the involvement of ecto‐protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.

Platelet activating factor (PAF) in memory formation: role as a retrograde messenger in long-term potentiation

Long-term potentiation (LTP) is a neurophysiological process that has been implicated in memory formation. The elevation of intracellular Ca2+ levels in postsynaptic neurons, an essential step in the induction of LTP in the hippocampus, can lead to activation of the enzyme acetyl-CoA:lyso-PAF acetyltransferase that is required for PAF synthesis in neurons. Thus, during the induction of LTP, stimulation of Ca2+ influx by glutamate receptors would lead to a postsynaptic increase in PAF biosynthesis. A main target for PAF action in neurons is the stimulation of neurotransmitter release via Ca(2+)-dependent vesicular exocytosis, a process that occurs presynaptically. In this article we describe the evidence obtained to-date for the pre- and postsynaptic events outlined, above, and demonstrate for the first time that during the induction of LTP by high-frequency stimulation (HFS) a 9-fold increase in PAF release to the extracellular environment occurs within 60 min following HFS. This finding provides the evidence that PAF can diffuse from postsynaptic sites of synthesis to presynaptic sites of action, and thus function as a retrograde messenger in the induction of LTP. Based on these data, we present a scheme in which postsynaptic glutamate receptors cooperate with presynaptic PAF receptors in a reverberating cycle that can amplify the transmission in a Hebbian synapse.

Elevated Plasma Level of Soluble F11 Receptor/Junctional Adhesion Molecule-A (F11R/JAM-A) in Hypertension

BACKGROUND The F11 receptor (F11R, also known as junctional adhesion molecule A (JAM-A)) plays a role in the development of hypertension in rat. Genetic variants in the human F11R gene were demonstrated to influence systolic blood pressure. In the present study, we investigated the relationship between F11R and hypertension by examining the levels of a circulating soluble form of F11R (sF11R) in hypertensive patients. METHODS Plasma sF11R was measured by enzyme-linked immunosorbent assay in 152 hypertensive and 166 normotensive subjects in whom seven tagging single-nucleotide polymorphisms (SNPs) in the F11R gene had been genotyped. RESULTS Plasma sF11R levels were significantly higher in hypertensive subjects than in normotensive subjects (median (interquartile) range): 162.8 (85.5-293.2) vs. 116.5 (74.1-194.8) pg/ml, P = 0.004), which remained significantly higher after adjusting for age, sex, body mass index (BMI), and homeostasis model assessment of insulin resistance (HOMA-IR) (P = 0.028). In stepwise multiple logistic regression, sF11R level (log-transformed) (P = 0.040), triglycerides (log-transformed) (P = 0.024), and HOMA-IR (log-transformed) (P < 0.001) were independently associated with hypertension. Plasma sF11R level correlated with systolic and diastolic blood pressures (r = 0.15, P < 0.001, and r = 0.13, P = 0.024, respectively). In stepwise multiple linear regression, hypertension (P = 0.013) and fibrinogen levels (P = 0.027) were significant independent predictors of sF11R level. A seven-locus haplotype, present in 2.1% of the subjects, was associated with higher sF11R level (P = 0.024). CONCLUSIONS These results further support a role of F11 receptor in the pathophysiology of human hypertension.