Endothelin converting enzyme-1-, endothelin-1-, and endothelin-3-like immunoreactivity in the rat brain.

Neurons likely to use endothelin as a neurotransmitter/neurohormone were mapped in the rat brain using polyclonal antibodies directed against endothelin-converting enzyme-1, endothelin-1, and endothelin-3. Anti-endothelin-converting enzyme-1 antibodies produced the most robust staining, permitting the best visualization of the distribution and morphology of neurons. Labeled neurons were found in the dorsal thalamic nuclei and reticular thalamic nuclei, medial preoptic area, pontine nucleus, and locus coeruleus. Localization of endothelin-converting enzyme-like immunoreactivity in the locus coeruleus and in the reticular nucleus of the thalamus suggests that endothelin is co-localized with norepinephrine and GABA, respectively. Additionally, endothelin-converting enzyme-like immunoreactivity was found in the globus pallidus, septal nuclei, and in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca, and the ventrolateral area of the caudate-putamen. Strong endothelin-converting enzyme-like immunoreactivity was found in a continuous band of pyramidal neurons throughout the neocortex primarily in layer V, extending into the cingulate gyrus and piriform cortex. Motor nuclei, including oculomotor, facial, and trigeminal nuclei, were also endothelin-converting enzyme-immunoreactive. In the cerebellum, Purkinje cells were stained. Non-neuronal cells such as oligodendroglia, microglia, and astrocytes generally were not endothelin-converting enzyme-immunoreactive, although astrocytes were rarely stained. Endothelin-converting enzyme-, endothelin-1-, and endothelin-3-like immunoreactivities were generally found co-existing in given nuclei. The diversity of neurons immunostained for endothelin suggests multiple roles of endothelin in the CNS.

7-Chlorokynurenate prevents NMDA-induced and kainate-induced striatal lesions.

NMDA-induced lesions of striatal cholinergic interneurons were attenuated by 7-chlorokynurenate (7-ClKyn), an antagonist of the glycine site of the NMDA receptor complex. However, it was not possible to demonstrate clearly that the mechanism of action of 7-ClKyn was in fact antagonism of the glycine site. Thus, the agonists at the glycine site, D-serine and 1-aminocyclopropane-1-carboxylic acid, failed to reverse the protection afforded by 7-ClKyn. Finally, 7-ClKyn also protected against lesions produced by kainate. The selectivity of 7-ClKyn under intracerebral administration is apparently insufficient for determining the role of the glycine site in NMDA-receptor mediated excitotoxicity.

Cerebral uric acid increases following experimental traumatic brain injury in rat.

Following traumatic brain injury (TBI), cortical and thalamic areas were analyzed histologically and by high-performance liquid chromatography with electrochemical detection for uric acid at various survival times. Following TBI, cortical uric acid was elevated by ten-fold at 24 and 48 h, but not at 1 h post-TBI. Histological evidence of neurodegeneration was found not only in cortex but also in the anteroventral thalamus. These data suggest that as in stroke, uric acid measurements may be a convenient and sensitive method for measuring peroxidative status in TBI.

Effects of putative cognition enhancers on the NMDA receptor by [3H]MK801 binding.

Piracetam, aniracetam, and D-cycloserine were tested for their ability to reduce inhibition of [3H]MK801 (dizocilpine) binding by 100 microM kynurenate. Piracetam (100 microM-1 mM) failed to reduce inhibition by kynurenate but stimulated [3H]MK801 binding in the absence of kynurenate. In contrast, D-cycloserine (30 microM-1 mM) and aniracetam markedly reduced this inhibition by kynurenate. Thus, cognition enhancers might function via at least some subtypes of NMDA receptors.

Rat striatum contains pure population of ETB receptors.

In most organs of the body, endothelin acts on endothelin ETA and ETB receptors that co-exist (albeit often on different cell types). Although virtually pure endothelin ETA receptors have been identified in some tissues (e.g., lung), no essentially pure endothelin ETB receptor tissue has been reported to date. [125I]Endothelin-1 bound to striatal membrane preparations with a Kd of 19.4 +/- 0.2 pM and Bmax of 496 +/- 8 fmol/mg protein. Endothelin-1 displaced [125I]endothelin-1 receptor binding with an IC50 of 23 pM. The endothelin ETB-selective antagonist BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma -methyl-leucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine) and agonist sarafotoxin 6C displaced [125I]endothelin-1 monophasically with IC50 values of 25 nM and 110 pM, respectively, whereas that of the endothelin ETA-selective antagonist BQ123 (cyclo(D-Trp-D-Asp-Pro-D-Val-Leu)) was 24 microM, values agreeing with cloned human endothelin ETB but not ETA receptors. Receptor autoradiography confirmed that rat striatum (but not white matter) contains essentially exclusively endothelin ETB receptors.

NMDA- and endothelin-1-induced increases in blood-brain barrier permeability quantitated with Lucifer yellow.

At 48 h following intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 nmol/microliter) or endothelin-1 (ET-1; 143 pmol/microliter), significant increases in brain penetration of the highly polar, fluorescent tracer Lucifer yellow were observed. The competitive NMDA receptor antagonist selfotel (CGS-19755; 30 nmol/microliter, i.c.) significantly reduced the NMDA-induced increases in blood-brain barrier permeability, but not those induced by ET-1. These results suggest that NMDA receptors can mediate increases in blood-brain barrier permeability but do not primarily mediate increases in blood-brain barrier permeability caused by ET-1. This is the first study to our knowledge investigating the relationship between excitotoxicity and disruption of the blood-brain barrier, a major pathophysiological event in stroke and traumatic brain injury.

A novel microdialysis probe designed for clinical use: potential analytical and therapeutic applications.

Significant obstacles to the use of microdialysis in the clinic for diagnostic or therapeutic purposes include the production of dedicated entry port through the skull and the formation of a tract by the insertion of a probe into the parenchyma. We have developed a microdialysis probe that is minimally invasive and can be combined with an intracranial pressure probe, recording electrode, or other intracranial probe, that is minimally invasive. Yet the surface area of this probe is very high, permitting high recovery efficiencies even at relatively high flow rates. This probe design makes possible minimally invasive measurement of the peroxidation product, uric acid, and excitatory amino acids, two analytes that increase in experimental traumatic brain injury in animals. Moreover, its large surface area makes therapeutic applications of microdialysis probes in the brain potentially feasible. A pilot evaluation of the ability of microdialysis to have therapeutic benefit in limiting experimental excitotoxin lesions induced in rat striatum by N-methyl-D-aspartate (NMDA) is reported.

Kinetics of 2-oxoglutarate uptake by synaptosomes from bovine and rat retina and cerebral cortex and regulation by glutamate and glutamine.

2-Oxoglutarate (2-OG) is a metabolic precursor of glutamate and may be utilized to replenish the neurotransmitter pool. 2-OG is rapidly transported into neurons by a high-affinity carrier that is particularly prevalent in glutamatergic terminals. Here we report the kinetics of [U-14C]2-OG uptake by crude synaptosomal preparations from bovine and rat retina and brain, and the modulatory effects of glutamate and glutamine. In all four tissues, 2-OG uptake was mediated by a high-affinity system (Kt approximately 1 microM) that was subject to negative feedback control by glutamate and biphasic modulation by glutamine (another precursor of neurotransmitter glutamate).