Vivian I. Teichberg

Enhancement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence.

Abstract The undecapeptide Substance P stimulates phagocytosis by mouse macrophages and human polymorphonuclear leukocytes. The activity of Substance P resides in its N-terminal tetrapeptide protion. Substance P and its N-terminal tetrapeptide are as active as tuftsin in their phagocytosis-stimulating activity and compete with tuftsin for its binding sites. The phagocytosis-enhancing activity of Substance P may play a role in inflammatory processes of neural origin where the involvement of the peptide has been implicated.

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin

The N‐methyl‐D‐aspartate receptor (NMDA‐R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post‐synaptic densities (PSDs). Since the activity of the NMDA‐R channel is dependent on the integrity of actin and leads to calpain‐mediated spectrin breakdown, we have investigated whether the actin‐binding spectrin may interact directly with NMDA‐Rs. Spectrin is reported here to interact selectively in vitro with the C‐terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA‐R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of α‐actinin‐2 and members of the PSD95/SAP90 family. The spectrin–NR2B interactions are antagonized by Ca2+ and fyn‐mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM‐dependent protein kinase II‐mediated NR2B phosphorylation. The spectrin–NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A‐ and C‐mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA‐R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.

A tetrameric subunit stoichiometry for a glutamate receptor-channel complex.

THE structure of glutamate receptor–channel (GluR) subunits has recently been shown to differ from that of other ligand-gated channels and to contain a voltage-gated channel-like pore-forming motif. The view that the structure of GluR complexes is similar to the pentameric structure of other ligand-gated channels was questioned here. Studies of the response properties of the GluR1 subunit of the AMPA subtype of GluRs, co-expressin Xenopus oocytes with its L646A mutant, which differs only by a greatly reduced sensitivity to quisqualate, provide new evidence suggesting that the GluR1 homomeric receptor channel has a tetrameric structure.

Blood-mediated scavenging of cerebrospinal fluid glutamate

The maintenance of brain extracellular glutamate (Glu) at levels below its excitotoxic threshold is performed by Glu transporters present on glia and neurons as well as on brain capillary endothelial cells which remove brain Glu into blood. The feasibility of accelerating the naturally occurring brain‐to‐blood Glu efflux was studied using paradigms based on the fate of Glu present in the cerebrospinal fluid or infused into the brain ventricles and monitored before, during, and after decreasing blood Glu levels with pyruvate and oxaloacetate, the respective Glu co‐substrates of the blood resident enzymes glutamate–pyruvate transaminase and glutamate–oxaloacetate transaminase. Results from cerebroventricular perfusions with [3H]Glu, intracerebroventricular injections of [3H]Glu, and measurements of the basal CSF Glu levels point out to the same conclusion that the intravenous administration of pyruvate and oxaloacetate which decreases blood Glu levels accelerates the brain‐to‐blood Glu efflux. We conclude that the brain extracellular Glu levels can be controlled in part by the blood Glu levels. The results may provide not only a rational explanation for the inhibition of Glu release and neuroprotective effects of parentally administered pyruvate in hemorrhagic shock and forebrain ischemia but could also outline a potential strategy for the removal of excess Glu in various neurodegenerative disorders.

Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by a post-proline cleaving enzyme from bovine brain

A post-proline cleaving enzyme isolated from bovine brain and previously shown to act on the neuropeptides thyrotropin releasing hormone (TRH: pGlu-His-Pro-NH2) and luteinizing hormone releasing hormone (LRH: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) has now been found to cleave the Pro4-Gln5 bond in substance P (SP: H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) to yield the N-terminal tetrapeptide H-Arg-Pro-Lys-Pro-OH and the C-terminal heptapeptide H-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. The site of cleavage of the undecapeptide was confirmed by chemical synthesis of SP peptide fragments, by high performance liquid chromatographic analysis, and by high-voltage paper electrophoresis, thin layer chromatography and amino acid analysis. In addition, SP was shown by enzyme kinetic analysis to act as a potent competitive inhibitor of the enzymatic hydrolysis of the synthetic post-proline cleaving enzyme substrate carbobenzoxy-Gly-Pro-para-nitroanilide. It is known that the heptapeptide is as active as SP itself in a variety of bioassays, whereas the tetrapeptide has been reported to induced cyclic AMP formation and neurite extension in neuroblastoma cells and to enehance the phagocytosis activity of macrophages. These observations suggest that SP may play a dual role, with one activity residing in its C-terminal part, and the other in the N-terminal part of the molecule.

Identification of the Amino Acid Subsets Accounting for the Ligand Binding Specificity of a Glutamate Receptor

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.

Homeostasis of glutamate in brain fluids: An accelerated brain-to-blood efflux of excess glutamate is produced by blood glutamate scavenging and offers protection from neuropathologies

L-Glutamate (Glu) homeostasis in brain extracellular fluids and its maintenance at low micromolar concentrations in the face of the extremely high Glu concentrations present in brain cells and synaptic vesicles have been commonly attributed to the very effective action of glutamate transporters present on neuronal and glial cells. This view however does not take into account the fact that the brain is highly vascularized and that the vasculature harbors a high density of glutamate transporters. In this article, we review the accumulated data establishing the existence of an efflux of excess Glu from brain extracellular fluids into blood. We describe plausible mechanisms accounting for this efflux and present evidence that the brain-to-blood Glu efflux is modulated by blood Glu levels and can be accelerated by blood Glu scavenging. The latter procedure shown here to afford brain neuroprotection in a rat model of closed head injury could be applicable, as a first-line therapy, in the various acute brain insults characterized by excess Glu in brain fluids.

Brain neuroprotection by scavenging blood glutamate

Excess glutamate in brain fluids characterizes acute brain insults such as traumatic brain injury and stroke. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As blood glutamate scavenging has been demonstrated to increase the efflux of excess glutamate from brain into blood, we tested the prediction that oxaloacetate-mediated blood glutamate scavenging causes neuroprotection in a pathological situation such as closed head injury (CHI), in which there is a well established deleterious increase of glutamate in brain fluids. We observed highly significant improvements of the neurological status of rats submitted to CHI following an intravenous treatment with 1 mmol oxaloacetate/100 g rat weight which decreases blood glutamate levels by 40%. No detectable therapeutic effect was obtained when rats were treated IV with 1 mmol oxaloacetate together with 1 mmol glutamate/100 g rat. The treatment with 0.005 mmol/100 g rat oxaloacetate was no more effective than saline but when it was combined with the intravenous administration of 0.14 nmol/100 g of recombinant glutamate-oxaloacetate transaminase, recovery was almost complete. Oxaloacetate provided neuroprotection when administered before CHI or at 60 min post CHI but not at 120 min post CHI. Since neurological recovery from CHI was highly correlated with the decrease of blood glutamate levels (r=0.89, P=0.001), we conclude that blood glutamate scavenging affords brain neuroprotection Blood glutamate scavenging may open now new therapeutic options.

The role of the N-terminal tetrapeptide in the histamine releasing action of substance P

Abstract Incubation of rat peritoneal mast cells with substance P, or with its N-terminal tetrapeptide segment but not with its C-terminal heptapeptide segment, caused a release of histamine. The half maximal release of histamine was observed with 8 × 10 −6 M substance P, while the N-terminal tetrapeptide was significantly less potent. The presence of the two basic amino acids in the N-terminal tetrapeptide was necessary but was not sufficient to account for the full histamine-releasing capacity of substance P. The inhibitory effect of different anti-inflammatory agents on mast cell degranulation caused by substance P and Compound 48/80 was compared.

Closing the gap between the in-vivo and in-vitro blood–brain barrier tightness

Numerous in-vitro models of the blood-brain barrier (BBB) have been developed in the hope to mimic as closely as possible the in-vivo BBB characteristics. Most models however display BBB tightness properties still very remote from those found in-vivo. We describe here the properties of an in-vitro BBB model in three configurations: primary porcine brain endothelial cells (PBEC) grown in a monoculture, or as a co-culture in close proximity to rat glial cells (contact), or with the latter at distance (non-contact). The BBB tightness as reflected by measurements of the permeability (Pe) to sucrose and of the transendothelial electrical resistance (TEER) showed that only the contact co-culture closely mimic the in-vivo BBB (Pe=0.1910(-6)+/-0.01 cm/s and TEER up to 1650 Omegacm2). While no changes in the expression pattern of three of the major tight junction proteins, claudin-5, occludin and ZO-1, were observed using immunohistochemistry, western blot analysis showed that the expression levels of claudin-5 and occludin increase when PBEC are cultured in contact with glial cells. In addition, we found, in the contact co-culture model, a reduced sensitivity of the TEER to vinblastine, a P-glycoprotein (Pgp) substrate that disrupts the cell cytoskeleton, indicating an improved functionality of the Pgp transporters in this configuration. We conclude that the close proximity of astrocytes is crucial to the development of a tight BBB.