dizocilpine-maleate and Cadaver

NMDA receptors in postmortem human spinal cord were analyzed using [3H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The average KD for [3H]MK-801 binding was 1.77 nM with a Bmax of 0.103 pmol/mg. The EC50 for stimulation of -3H-MK-801 binding with L-glutamate was 0.34 microM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.

Topics: Cadaver; Cell Membrane; Dizocilpine Maleate; Glutamic Acid; Humans; Kinetics; Postmortem Changes; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Tritium

Polyamines are thought to modulate the activation of NMDA receptors through a unique allosteric regulatory site. The effects of polyamines on the binding of [3H]MK-801 were measured in cortical and hippocampal tissue surgically removed from patients with temporal lobe epilepsy (TLE). The polyamine agonist spermidine increased the binding of [3H]MK-801 in the cortex in a dose-dependent manner and this effect could be blocked by the weak partial agonist diethylenetriamine (DET). Spermidine decreased the Kd of [3H]MK-801 for the NMDA receptor but did not alter the density of receptors. Spermidine had essentially the same effect on Kd and Bmax measured in the dentate gyrus of TLE subjects and the cortex and dentate gyrus of postmortem controls. Moreover, there was no difference in the density of binding sites between postmortem and TLE subjects in either region. The binding of [3H]MK-801 in human cortex was decreased by 30% by incubation with DET or by prewashing the tissue sections. In contrast, DET did not alter the binding of [3H]MK-801 in rat cortex and prewashing sections produced an increase rather than a decrease in binding. These results suggest that there are different endogenous modulators for the polyamine site in rat and human tissue. The inverse agonist 1,10-diaminodecane decreased the binding of [3H]MK-801 in a dose-dependent manner. These results suggest that the fundamental modulatory properties of polyamines in rat and human tissues are essentially the same and that endogenous polyamines may regulate human NMDA receptors.

Topics: Adult; Animals; Brain; Cadaver; Cerebral Cortex; Diamines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Female; Hippocampus; Humans; Male; Middle Aged; Polyamines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine

[3H]MK-801 binding was used as an index of the glutamate receptor N-methyl-D-aspartate-subtype channel to examine the influence of gender, age, mode of death (agonal status), interval between death and autopsy (postmortem delay), and time in storage at -70 degrees C in well washed homogenate preparations from postmortem human frontal cortex. Basal binding and the modulatory effects of glutamate, glycine, spermidine, and zinc were examined with respect to these variables. Basal binding was sensitive to agonal status, being higher in sudden death cases. The effect of added glutamate and glycine was sensitive to age, with a trend toward lower binding with increasing age. The effect of added spermidine alone was sensitive to storage time at -70 degrees C, the binding being higher with longer storage time. The effect of added zinc was also sensitive to postmortem delay, with zinc causing a greater reduction in binding with shorter postmortem delays. Thus, with the exception of gender, all variables examined influenced [3H]MK-801 binding, highlighting the attention that should be given to these factors in postmortem studies in normal and diseased human subjects.

Topics: Aged; Aged, 80 and over; Analysis of Variance; Cadaver; Cerebral Cortex; Dizocilpine Maleate; Female; Glutamates; Glutamic Acid; Glycine; Humans; Male; Middle Aged; Spermidine; Time Factors; Tritium; Zinc