Rapid transferrin efflux from brain to blood across the blood-brain barrier

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. Whereas high-molecular-weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5-fold faster than the efflux of holo-transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood-to-brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.     

Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander

Light stimulates dopamine release in the retina and has been shown to rapidly up-regulate rod opsin mRNA. In the present study, we tested the effect of dopamine on rod opsin mRNA expression and examined the hypothesis that dopamine can mediate a light-evoked increase in opsin gene expression. Northern blots showed that a 30-min light-exposure increased rod opsin mRNA expression 27%. In situ hybridization on isolated rods showed that 500 nM dopamine and 1 microM quinpirole (dopamine D2/D3/D4 agonist) increased opsin mRNA 45% and 26%, respectively. The effect of quinpirole was selectively blocked by the D4 antagonist, L750,667 (20 microM). In very low density cultures, quinpirole increased opsin expression 46%, suggesting a direct effect on rod photoreceptors. Consistent with a dopamine D4 receptor mechanism, 1 microM H-89 (protein kinase A inhibitor) increased opsin mRNA 39%. Finally, intravitreal injection of quinpirole increased opsin mRNA 21% whereas injection of L750,667 (10 microM) blocked the light-evoked increase in opsin expression. These data show that rod opsin mRNA is up-regulated by dopamine binding a D4-like receptor on rods, possibly through inhibition of protein kinase A, and that endogenous dopamine can mediate the light-evoked increase in opsin mRNA expression.     

Synthesis and BZR affinity of pyrazolo[1,5-a]pyrimidine derivatives. Part 1: study of the structural features for BZR recognition

Examination of the pharmacophoric points of the pyrazolo[1,5-a]pyrimidine derivatives, ligands for BZR, previously published led us to the design of a novel class of 3,6-diaryl-4,7-dihydro-pyrazolo[1,5-a]pyrimidin-7-ones and to determine the groups involved in the BZR recognition.     

Differential effect of dopamine catabolism and uptake inhibition on dopamine-induced calcium dysregulation and viability loss

The present study was aimed at evaluating of the effects of dopamine (DA) toxicity on PC12 cells' calcium homeostasis, cellular viability, and free radical levels. Moreover, the effect of receptor inhibition, and DA metabolism and reuptake antagonism on all parameters was also evaluated. Acute treatment with DA impaired the ability of PC12 cells to buffer excess calcium after K+-depolarization, decreased cellular viability by approximately 35%, and increased free radical levels by about 10% in a dose dependent manner. Pretreatment with both active and inactive pargyl monoamine oxidase inhibitors (MAOi) protected PC12 cells from DA toxicity on cellular viability and free radical levels, regardless of the presence or absence of their target enzymes in PC12 cells. These results suggest a lack of specific involvement of DA metabolism by MAO in dopamine's effects on cellular viability and production of free radicals. However, DA-induced dysregulation of calcium homeostasis seems to be more specifically mediated by DA metabolism by MAO. Results indicate that, in order for toxicity to occur the DA must be taken up into the cells. DA receptors do not mediate dopamine cytoxicity, and the D2 receptor plays a modest role in DA-induced calcium dysregulation and generation of free radicals. Moreover, DA-induced cell viability loss is not mediated by calcium, nor by caspase-3 enzyme, but is prevented by inhibition of mitochondrial permeability transition pores.