Characterization of desensitization in recombinant N-methyl-D-aspartate receptors: comparison with native receptors in cultured hippocampal neurons.

In the present study we have characterized the effect of Ca2+, glycine, and agonist concentration on inactivation and desensitization in native and recombinant N-methyl-d-aspartate (NMDA) receptors. In agreement with earlier studies on neurons, we found that in the presence of saturating glycine concentrations, lowering [Ca2+]o, will decrease inactivation of NMDA receptors in cultured hippocampal neurons. However, unlike native NMDA receptors under the same recording conditions, recombinant receptors did not exhibit Ca2+-dependent inactivation. We also show that the glycine-insensitive desensitization observed in the recombinant receptors is subunit dependent, as NR1a2A and NR1a2B receptors significantly desensitized while the NR1a2C combination did not. Furthermore, we show this form of desensitization in NR1a2A receptors is due to classic agonist-induced desensitization. In addition, we demonstrate the presence of glycine-dependent desensitization in recombinant receptors. The ability of glycine to inhibit desensitization correlates to the rank order of glycine's affinity for potentiating the peak response for each subtype. Finally, using ifenprodil in the presence of high and low glycine concentrations, we present evidence that both 2A-like and 2B-like subtypes of receptors can independently coexist in single neurons.

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Characterization of glutamate binding sites in receptors assembled from transfected NMDA receptor subunits.

Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using L-[3H]glutamate and [3H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg2+ can alter [3H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [3H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [3H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.

The influence of vasovasostomy on antisperm antibodies in rats.

Serum antisperm antibodies were studied in Sprague-Dawley rats after vasectomy and vasovasostomy. Animals received a bilateral vasectomy, a vasectomy followed 3 mo later by vasovasostomy, or sham operations. Blood samples were obtained at 1, 3, 4, and 7 mo, and antisperm antibodies were assayed by an enzyme-linked immunosorbent assay. After vasectomy reversal was performed at 3 mo, antisperm antibodies were significantly higher in rats in the vasovasostomy group at 4 mo than in animals that had a persisting vasectomy or sham operations. At 7 mo, the antisperm antibody level for the vasovasostomy group was approximately double that for the vasectomized rats. Spermatic granulomas occurred in 76% of rats after vasovasostomy. Antisperm antibody levels were higher in vasovasostomized animals with granulomas than in those lacking granulomas. The results suggest that vasovasostomy may stimulate an antibody response to sperm rather than lead to a reduced response, as was anticipated upon removal of the obstruction. Spermatic granulomas may serve as sires for continued antigenic challenge. The observed increase in antisperm antibodies after vasovasostomy in Sprague-Dawley rats may be related to their relatively low immunologic responsiveness to vasectomy, with vasovasostomy serving as a second major immunologic challenge, aided by the formation of an additional granuloma. In the more responsive Lewis strain, we previously observed a rise in antisperm antibodies after the initial vasectomy, with no further increase after vasovasostomy.

Testicular alterations are linked to the presence of elevated antisperm antibodies in Sprague-Dawley rats after vasectomy and vasovasostomy.

The relationship between alterations in testicular histology and antisperm antibodies was studied after vasectomy and vasovasostomy in Sprague-Dawley rats, which are immunologically relatively non-responsive to vasectomy. Testes were prepared for histologic study at intervals up to seven months after vasectomy, vasectomy followed three months later by vasovasostomy, or sham operations. Antisperm antibodies were assessed with an ELISA. Testicular alterations, which were observed in a minority of animals after vasovasostomy, consisted mainly of depletion of germ cells. Mean serum antisperm antibody levels were greater for animals with altered testes than for rats with normal testicular histology. In addition, the proportion of rats that showed a positive antisperm antibody response was greater among animals with testicular changes than among those with unaltered testes. When the present results on Sprague-Dawley rats were compared with previous findings on the highly responsive Lewis strain, it was evident that the incidence of testicular changes and the proportion of positive antibody responders were greater in the Lewis strain. However, elevated antisperm antibodies and testicular alterations appeared to be more tightly linked in the less responsive Sprague-Dawley rats.