James K. T. Wang

Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes

The wide‐ranging neuronal actions of glutamate arc thought to be mediated by postsynaptic N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non‐NMDA agonists in the presence of Mg2+, results in Ca2+‐depeodent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non‐NMDA agonists is blocked by a non‐NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non‐NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important rote in transmitter interactions in die striatum.

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

Abstract: The wide‐ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non‐NMDA agonists, in a concentration‐dependent manner, resulted in Ca2+‐dependent release of noradrenaline from vesicular transmitter stores. The NMDA‐stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non‐NMDA agonists was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non‐NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.

Presynaptic NMDA Receptors Display Physiological Characteristics of Homomeric Complexes of NR1 Subunits that Contain the Exon 5 Insert in the N‐Terminal Domain

Abstract: The subunit composition of the N‐methyl‐d‐aspartate (NMDA) glutamate receptor affects both its channel activity and its sensitivity to modulation by a wide variety of substances. Expression studies in oocytes and physiological studies in neurons indicate that endogenous postsynaptic NMDA receptors are heterooligomeric complexes of NR1 and NR2 subunits. To deduce the subunit composition of the presynaptic NMDA receptor on noradrenergic nerve terminals, we examined the modulation of NMDA‐evoked norepinephrine (NE) release from hippocampal synaptosomes. At high glycine concentrations, the NMDA‐evoked release was not potentiated by reducing reagents, low micromolar Zn2+ or Ni2+, polyamines, or 100 µM histamine. It was also not inhibited by oxidizing agents or physiological concentrations of protons but was inhibited by high micromolar Co2+, Zn2+, and Ni2+, but not Fe3+, by high micromolar ifenprodil, and by 1 mM histamine. At low glycine concentrations, it was potentiated by spermine. These characteristics are similar to those displayed by homooligomeric complexes of NR1 subunits that contain in the N‐terminal domain the 21‐amino‐acid insert encoded by exon 5. These data provide physiological evidence that some endogenous NMDA receptor complexes may contain only the NR1 (+ exon 5) subunits.

Neurotrophin-3 mediates the autocrine survival of the catecholaminergic CAD CNS neuronal cell line

The mechanisms for neuronal survival in the CNS are not well understood, but are likely to be complex due to possible autocrine and redundant neurotrophic support. Most studies have focused on the nerve growth factor (NGF)/TrkA pathway in peripheral neurons, and little is known regarding the other neurotrophins, particularly neurotrophin‐3 (NT3)/TrkC. Progress has also been hampered by the paucity of homogenous and accessible CNS neuronal experimental models. We now report that the novel catecholaminergic CNS cell line, CAD, is capable of autocrine survival mediated by NT3. The CAD cell is of CNS neuronal origin and can survive and morphologically differentiate in the absence of exogenously provided trophic factors. However, neutralizing reagents against NT3 (the neutralizing TrkC‐IgG fusion protein and anti‐NT3 antibodies), but not those that block the other neurotrophins, inhibited survival of differentiating CAD cells. Moreover, Trk phosphorylation was detected in CAD cells and its inhibition by K252a was correlated with K252a‐induced apoptosis. Finally, endogenous NT3 was detectable in CAD cell extracts by a specific ELISA assay. Thus, CAD cells possess an autocrine survival capability mediated by NT3, and may provide a valuable model system for studying the signaling pathways that mediate the actions of this little understood neurotrophin.

Involvement of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth induced by cAMP treatment and serum deprivation in a central nervous system cell line, CAD derived from rat brain.

A central nervous system (CNS) cell line, CAD, is known to differentiate in the absence of serum. This cell line was found to differentiate by the treatment of cAMP. Expression of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) was induced to about 2-fold or more on day 1, and was continued at a high level for 5 days after the exposure to differentiating conditions. Neurite extension was stimulated from day 1 and continued for 5 days, suggesting that CaM kinase II activity is correlated with neurite outgrowth. Of the four distinct isoforms (alpha, beta, gamma, and delta) of the kinase, the delta isoform was the major isoform in CAD cells. The splicing pattern of this isoform in the differentiated cells differed from that in undifferentiated cells, suggesting that expression of CaM kinase II is regulated during neural differentiation.

Cu2+ induces Ca2+-dependent neurotransmitter release from brain catecholaminergic nerve terminals.

CuCl2, ZnCl2 and NiCl2, but not CdCl2 or CoCl2, induced transmitter release from superfused rat hippocampal and striatal synaptosomes preloaded with, respectively, [3H]noradrenaline and [3H]dopamine. Cu2+ was the most potent and effective, acting in a concentration- (0.1-300 microM) and time-dependent (peak effect occurring at 2-3 min) manner. The amount of Cu2+-induced release over a 5 min period is similar to that induced by depolarization with high KCl or the K+ channel blocker 4-aminopyridine. However, the time course of the Cu2+-induced release is slower and the effect of Cu2+ is not reversed by washout. Cu2+-induced catecholamine release requires extracellular calcium (Ca2+) and is inhibited by the Ca2+ channel blocker Cd2+, and in the case of noradrenaline, by the voltage-gated Na+ channel blocker tetrodotoxin. The ability of Cu2+ to induce massive Ca2+-dependent transmitter release from brain catecholaminergic nerve terminals may contribute to the neuropathological processes associated with Cu2+ toxicity in Wilsons disease.

CNS voltage-dependent Na+ channel expression and distribution in an undifferentiated and differentiated CNS cell line

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channels structural differences play a role in channel distribution.

Increased expression in human astrocytomas of a 100 kDa protein with sequence homology to the ros tyrosine kinase domain.

A monoclonal antibody against the v-ros synthetic peptide VWETLTLGQQPYPGLSN IEVL (amino acid residues 455-475 of v-ros), which is in the conserved region of the c-ros tyrosine kinase domain, was used for Western blotting of human astrocytoma specimens. High levels of a 100 kDa protein were detected in many of these primary brain tumours. In contrast, low levels of this 100 kDa protein were consistently found in the nontumour brain control samples. This 100 kDa protein is, however, probably not the c-ros protein, but may be a novel cytosolic protein-tyrosine kinase and a marker for early transformation of astrocytomas.