Interaction of the N-methyl-D-aspartic acid receptor NR2D subunit with the c-Abl tyrosine kinase.

The COOH-terminal domain of the NR2D subunit of the NMDA receptor contains proline-rich regions that show striking homology to sequences known to bind to Src homology 3 (SH3) domains. To determine whether the proline-rich region of the NR2D subunit interacts with specific SH3 domains, in vitro SH3 domain binding assays were performed. A proline-rich fragment of the NR2D subunit (2D(866-1064)) bound to the Abl SH3 domain but not to the SH3 domains from Src, Fyn, Grb2, GAP, or phospholipase C-gamma (PLCgamma). Co-immunoprecipitation of NR2D with Abl suggests stable association of NR2D and Abl in transfected cells. The SH3 domain plays an important role in the negative regulation of Abl kinase activity. To determine whether the interaction of NR2D with the Abl SH3 domain alters Abl kinase activity, Abl was expressed alone or with NR2D in 293T cells. Autophosphorylation of Abl was readily observed when Abl was expressed alone. However, co-expression of Abl with 2D(866-1064) or full-length NR2D inhibited autophosphorylation. 2D(866-1064) did not inhibit DeltaSH3 Abl, indicating a requirement for the Abl SH3 domain in the inhibitory effect. Similarly, 2D(866-1064) did not inhibit the catalytic activity of Abl-PP, which contains two point mutations in the SH2-kinase linker domain that release the negative kinase regulation by the SH3 domain. In contrast, the full-length NR2D subunit partially inhibited the autokinase activity of both DeltaSH3 Abl and Abl-PP, suggesting that NR2D and Abl may interact at multiple sites. Taken together, the data in this report provide the first evidence for a novel inhibitory interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase.

Methylation of the NMDA receptor agonist L-trans-2,3-pyrrolidine-dicarboxylate: enhanced excitotoxic potency and selectivity.

This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring of L-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increased in vivo excitotoxic potency 1000-fold. Injections (1 mu 1) in rat dorsal hippocampus of cis- and trans-5-methyl-L-trans-2,3-PDC (0.1 nmol) induced 50-70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol of L-trans-2,3-PDC. Further, cis- and trans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-L-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. or example, the activity of the compounds as inhibitors of 3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50 values: 5, 49, and 16 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), and in vitro excitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA excitotoxins, cis- and trans-5-methyl-L-trans-2,3-PDC will be of value studying excitotoxic mechanisms, MDA-receptor-mediated pathology, and NMDA receptor heterogeneity.

Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes.

The pharmacology of recombinant NR1a/NR2D NMDA receptors expressed in Xenopus oocytes was examined and compared to the pharmacology of NR1a/NR2A, NR1a/NR2B and NR1a/NR2C heteromers. The NR1/NR2D heteromer showed a pharmacological profile distinct from each of the other NR1/NR2 heteromers. This unique pharmacological profile was characterized by a relatively lower affinity for the agonist homoquinolinate and the antagonists 2-amino-5-phosphonopentanoate (D-AP5) and (R,E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPPene) but not for the antagonists (+/-)-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid (PBPD) and alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid (EAB515). NR2D-containing receptors displayed a pharmacological profile most similar to that observed for receptors containing the genetically related NR2C subunit. These findings parallel observations obtained for native NMDA receptors in the medial thalamus (presumed to contain NR2D subunits) and forebrain (presumed to contain NR2A and NR2B subunits). Thus, only compounds that discriminate between either NR2A- or NR2B-containing heteromers and NR2D-containing heteromers also discriminate between forebrain and medial thalamic NMDA receptors. While the pharmacology of the NR1a/NR2D receptor shows many parallels to the medial thalamic NMDA receptor, some differences were observed. Certain compounds which discriminate between medial thalamic and cerebellar (presumed to contain NR2C subunits) receptors (e.g., homoquinolinate, D-CPPene) did not show a similar selectivity for NR1a/NR2D receptors relative to NR1/NR2C receptors. Co-expression of NR1a, NR2B and NR2D subunits in Xenopus oocytes resulted in the formation of heteromeric complexes with unique pharmacological properties, suggesting the co-existence of these two distinct NR2 subunits in the same receptor complex.

Glycine modulates ethanol inhibition of heteromeric N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Ethanol inhibits N-methyl-D-aspartate (NMDA) receptor-mediated responses at pharmacologically relevant concentrations, suggesting that inhibition of NMDA receptors may underlie some of the actions of ethanol in the central nervous system. We examined the ability of glycine to modulate ethanol inhibition of four recombinant heteromeric NMDA receptors (NR1a/NR2A through NR2D) expressed in Xenopus oocytes. Ethanol dose-response analysis revealed enhanced inhibitory efficacy of ethanol in the presence of subsaturating glycine concentrations at the NR1/NR2A, NR1/NR2C, and NR1/NR2D receptors. When assayed over a range of glycine concentrations, ethanol exhibited both glycine-reversible and glycine-independent inhibition of NMDA receptors. In contrast, ethanol inhibition of recombinant NMDA receptors was independent of NMDA concentration. Glycine reversal of ethanol inhibition suggested that ethanol might lower the affinity of glycine for the NMDA receptor and thereby decrease response magnitude. Consistent with this hypothesis, ethanol significantly reduced glycine affinity at NR1/NR2A and NR1/NR2C receptors. Evaluation of the glycine-independent component of ethanol inhibition demonstrated that in the presence of saturating concentrations of glycine, the NR1/NR2A and NR1/NR2B receptors were more sensitive to ethanol than the NR1/NR2C and NR1/NR2D receptors. Activation of the NR1/NR2D heteromers by NMDA and low concentrations of glycine elicited responses characterized by an initial peak followed by a lower-amplitude plateau response, which is consistent with glycine-sensitive desensitization as previously described for native NMDA receptors. In addition, nondesensitizing NR1/NR2B responses elicited in the presence of subsaturating concentrations of glycine were frequently converted into desensitizing responses by the addition of ethanol, an effect that was reversed with increasing glycine concentrations. The ability of ethanol to promote glycine-sensitive desensitization further suggests an interaction between glycine and ethanol inhibition of the NMDA receptor. Taken together, the results of the present report demonstrate that ethanol inhibition of NMDA receptors has both glycine-reversible and glycine-independent components, suggesting two distinct molecular mechanisms for ethanol inhibition of NMDA receptors.

Site-directed mutagenesis of N-linked glycosylation sites on the gamma-aminobutyric acid type A receptor alpha 1 subunit.

Oligonucleotide-directed mutagenesis was used to mutate the two potential sites for N-linked glycosylation on the rat gamma-aminobutyric acid (GABA)A receptor alpha 1 subunit. Wild-type (WT) or mutant alpha 1 subunits [asparagine to glutamine substitutions at position 10 (alpha 1Q10), 110 (alpha 1Q110), or both 10 and 110 (alpha 1Q10/110)] were coexpressed with beta 1 and gamma 2 subunits in Xenopus oocytes. Removal of either one or both potential sites for N-linked glycosylation resulted in expression, in Xenopus oocytes, of functional GABAA receptors with pharmacological properties similar to those observed for the WT receptor. WT and mutant alpha 1 subunits were co-transfected with beta 1 and gamma 2 subunits in human embryonic kidney 293 cells. WT and mutant alpha 1 subunits expressed in 293 cells were photoaffinity labeled with [3H]flunitrazepam. Co-transfection of alpha 1WT, alpha 1Q10, or alpha 1Q110 subunits in combination with beta 1 and gamma 2 GABAA receptor subunits resulted in the labeling of single bands, with approximate molecular masses of 54, 49, and 50 kDa, respectively. The decrease in molecular mass for both the alpha 1Q10 and alpha 1Q110 mutants suggests that both consensus sequences for N-linked glycosylation are used in 293 cells. Low levels of [3H]flunitrazepam binding prevented visualization of the alpha 1Q10/110 double mutant. The 293 cells transfected with either the alpha 1Q10 or alpha 1Q110 mutant in combination with beta 1 and gamma 2 subunits expressed significantly lower levels of [3H]Ro15-1788 binding, relative to WT levels. In addition, [3H]Ro15-1788 binding was undetectable in 293 cells expressing the alpha 1Q10/110 double mutant. When transfected 293 cells were grown at 30 zero, [3H]Ro15-1788 binding to alpha 1Q10 and alpha 1Q110 GABAA receptors was restored to levels comparable to that for WT receptors. [3H]Ro15-1788 binding to alpha 1Q10/110 was not reliably detected at 30 zero. Similar results were observed using [3H]muscimol. These data suggest that intracellular processing and transport of the glycosylation-deficient GABAA receptor alpha 1 subunit is temperature sensitive. Furthermore, the observed differences between the two expression systems may be accounted for by the typically lower temperature used for maintaining microinjected Xenopus oocytes. Thus, although glycosylation is not an absolute requirement for GABAA receptor expression, it has a profound effect on the processing of at least the alpha 1 receptor and its subsequent assembly into a mature receptor.

The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition.

The relationship between four pharmacologically distinct NMDA receptor subtypes, identified in radioligand binding studies, and the recently identified NMDA receptor subunits (NR1a-g, NR2A-D) has not been determined. In this report, we demonstrate that the anatomical distribution of the four NMDA receptor subtypes strikingly parallels the distribution of mRNA encoding NR2A-D subunits. The distribution of NR2A mRNA was very similar to that of "antagonist-preferring" NMDA receptors [defined by high-affinity 3H-2-carboxypiperazine-4-yl-propyl-1-phosphonic (3H-CPP) binding sites; correlation coefficient = 0.88]. Agonist-preferring NMDA receptors localized to brain regions expressing both NR2B mRNA and NR1- mRNA (NR1 splice variant lacking insert 1). NR2C mRNA was largely restricted to the cerebellar granule cell layer, a region that displays a unique pharmacological profile. NR2D mRNA localized exclusively to those diencephalic nuclei that have a fourth, distinct pharmacological profile (typified by the midline thalamic nuclei). The pharmacology of native NMDA receptors was compared to that of heteromeric NMDA receptors expressed in Xenopus oocytes (NR1/NR2A, NR1/NR2B, NR1/NR2C). The oocyte-expressed NR1/NR2A receptor displayed a higher affinity for antagonists and a slightly lower affinity for agonists than the NR1/NR2B receptor. These patterns are analogous to those found for radioligand binding to native receptors in the lateral thalamus and medial striatum, respectively. NMDA receptors in the lateral thalamus (with a high density of NR2A subunit mRNA) displayed higher affinity for antagonists and a lower affinity for agonists than did NMDA receptors of the medial striatum (a region rich in NR2B mRNA). Relative to the NR1/NR2A and NR1/NR2B receptors, oocyte-expressed NR1/NR2C receptors had a lower affinity specifically for both D-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (D-CPPene) and homoquinolinate (HQ). This pattern was identical to that observed for cerebellar (NR2C-containing) versus forebrain (NR2A- and NR2B-containing) NMDA receptors. Taken together, the data in this report suggest that the four previously identified native NMDA receptor subtypes differ in their NR2 composition. Furthermore, the NR2 subunits significantly contribute to the anatomical and pharmacological diversity of NMDA receptor subtypes.

Altered patterns of N-linked glycosylation of the Torpedo acetylcholine receptor expressed in Xenopus oocytes.

The nicotinic acetylcholine receptor (AChR) from Torpedo electroplax is an oligomeric transmembrane glycoprotein made up of four highly homologous subunits in a stoichiometry of alpha 2 beta gamma delta. The role of N-linked glycosylation of the AChR has been studied in several cell lines and these studies have suggested that the addition of carbohydrate may be important for receptor expression. While Xenopus oocytes have proven to be an invaluable tool for studying the AChR, little is known about N-linked glycosylation of the oocyte-expressed receptor. The present report demonstrates that the oocyte-expressed AChR is glycosylated and contains the same number of oligosaccharide residues per subunit as the native receptor. However, unlike the native Torpedo receptor which contains both high mannose and complex oligosaccharides, the oocyte-expressed AChR contains only high mannose oligosaccharide modifications. However, as has been well documented, the Torpedo AChR expressed in oocytes is fully functional, demonstrating that the precise nature of the oligosaccharide modification is not critical for receptor function. The role of the oligosaccharide component of the AChR in receptor function was examined using tunicamycin (TM) to inhibit N-linked protein glycosylation. TM treatment resulted in a 70-80% inhibition of AChR expression in oocytes. Functional, unglycosylated receptors were not expressed; receptors expressed in TM-treated oocytes were functional wild-type, glycosylated AChR, formed only during the initial 12 hr of TM exposure. These data suggest that while glycosylation of the oocyte-expressed Torpedo AChR is required for assembly of subunits into a functional receptor, as has been demonstrated in other cells, oocyte modification of normal Torpedo glycosylation patterns does not affect receptor function or assembly.

Functional acetylcholine receptors expressed in Xenopus oocytes after injection of Torpedo beta, gamma, and delta subunit RNAs are a consequence of endogenous oocyte gene expression.

The nicotinic acetylcholine receptor (AChR) is an oligomeric transmembrane glycoprotein consisting of four homologous subunits in a stoichiometry alpha 2 beta gamma delta. Xenopus oocytes were used to study the effects of selectively deleting the alpha subunit of the Torpedo californica AChR on functional receptor expression. Oocytes microinjected with only Torpedo beta, gamma, and delta subunit RNAs showed small acetylcholine-elicited currents. These "alpha-less" AChRs were pharmacologically similar to the wild-type (i.e., Torpedo alpha 2 beta gamma delta) receptor. Actinomycin D, which blocks endogenous RNA transcription, completely inhibited the expression of alpha-less but not wild-type receptor. Coinjection of antisense RNA to the alpha subunit of the Xenopus muscle AChR with Torpedo beta, gamma, and delta subunit RNAs significantly reduced expression of the alpha-less AChRs without altering expression of wild-type receptors. These results indicate that Xenopus oocytes express low levels of AChR alpha subunit mRNA that, when translated, can lead to the formation of functional Xenopus-Torpedo AChR hybrids. These results strongly suggest that, unless the potential contribution of endogenous subunits can be determined, caution must be exercised when analyzing the effects of subunit deletions on multisubunit protein expression in Xenopus oocytes.

Characterization of dihydrodiol dehydrogenase in rat H-4IIe hepatoma cells.

Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP+-dependent oxidation of a variety of trans-dihydrodiol proximate carcinogens, a reaction that may suppress their carcinogenicity. Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a substrate, this enzyme can be detected spectrophotometrically in rat H-4IIe hepatoma cells with a specific activity similar to that observed in rat liver cytosol. The hepatoma cell enzyme is potently inhibited by 6-medroxy-progesterone acetate (IC50 = 38 nM) and indomethacin (IC50 = 3.5 microM). These cells contain 3 alpha-hydroxysteroid dehydrogenase which is also sensitive to inhibition by the same two drugs. Chromatofocusing of hepatoma cell lysates indicates that both dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities coelute with a pI = 5.8. Western blot analysis of hepatoma cell lysates, using rabbit anti-rat 3 alpha-hydroxy-steroid/dihydrodiol dehydrogenase serum detects a single immunoreactive species with a Mr 34,000. Using this antiserum it was possible to immunotitrate both these enzyme activities in H-4IIe lysates. Exposure of confluent cells to either 10 microM benz[a]anthracene or 10 microM dexamethasone, which are known inducers in H-4IIe cells of aryl-hydrocarbon hydroxylase and tyrosine aminotransferase respectively, failed to elevate dihydrodiol dehydrogenase activity. The following agents also failed to induce dihydrodiol dehydrogenase activity: phenobarbital, ethoxyquin, phenolic anti-oxidants, testosterone, estradiol-17 beta, and growth hormone. Since the hepatoma cell enzyme has properties in common with the purified rat liver enzyme (which is identical to 3 alpha-hydroxysteroid dehydrogenase) including, Mr, pI, immunoreactivity, and sensitivity to drug inhibition, this cell line represents a useful system for studying the role of dihydrodiol dehydrogenase in the further metabolism of trans-dihydrodiols. Interestingly, the enzyme does not appear to be under the control of known inducers of phase I and phase II drug metabolizing enzymes.

Control of Torpedo acetylcholine receptor biosynthesis in Xenopus oocytes.

The nicotinic acetylcholine receptor (AcChoR) of Torpedo electroplax is a multisubunit transmembrane glycoprotein complex with a subunit stoichiometry of alpha 2 beta gamma delta. The RNAs for the separate subunits were transcribed in vitro from cDNAs inserted in pSP64T vectors and microinjected in Xenopus oocytes. Microinjected in vitro-transcribed RNAs were stable, with a half-life of 72 hr. Xenopus oocytes assembled functional AcChoRs from the subunit-specific RNAs. These receptors were inserted in the cell membrane and could be detected as early as 6 hr after RNA microinjection. The oocyte-expressed AcChoR subunits could be immunoprecipitated with anti-Torpedo AcChoR subunit antibodies. Expression of the AcChoR in oocytes proceeded linearly for 72 hr after microinjection. While the amount of RNA injected did not alter the linearity of the expression time course, the rate of receptor expression in oocytes showed a saturable dependence on RNA concentration. Varying the relative amount of alpha-subunit RNA microinjected into oocytes had a striking effect on receptor expression. This effect was specific for the alpha-subunit. These results suggest that transcript availability may control receptor expression in Xenopus oocytes. In addition, the availability of the alpha-subunit may be a limiting factor for receptor expression.

Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines.

Tumor cell resistance to alkylating agents was studied by examining Walker 256 rat mammary carcinoma cells differentially sensitive to nitrogen mustards. A resistant subpopulation (WR) was selected by exposure to chlorambucil. WR cells showed approximately a 15-fold resistance to the cytotoxic effects of nitrogen mustards and elevated glutathione S-transferase (GST) activity when compared to the sensitive parent cell line (WS). To extend these findings, the GSTs from WR and WS were purified by affinity chromatography on S-hexylglutathione coupled to epoxy-activated agarose. Substrate specificity experiments using purified GSTs demonstrated different profiles of enzyme activity for WR and WS and suggested differential isoenzyme expression in these two cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that the major GST present in both WR and WS was a 26,000-Da subunit that was immunologically distinct from the rat liver GSTs. This GST subunit cross-reacted with antibodies against anionic human placental GST. In addition, three GST forms common to rat liver (29,500, 28,500 and 27,500 molecular weight) were also identified. Overexpression of the 29,500-Da protein was observed in WR cells. These data suggest that differential expression of GST subunits may contribute to the nitrogen mustard-resistant phenotype.

Central respiratory stimulation produced by thyrotropin-releasing hormone in the cat.

Thyrotropin-releasing hormone (TRH) was administered intracerebroventricularly and it's effects on respiration were evaluated in the alpha-chloralose anesthetized cat. Respiratory activity was measured using a Fleisch pneumotachograph to monitor tracheal airflow. TRH (0.28-28 nmol) caused an elevation in respiratory minute volume which was due to an increase in respiratory rate with no effect on tidal volume. The site of TRH-induced tachypnea was in the hindbrain as both injections into the cisterna magna and the fourth ventricle produced similar effects. No changes in respiratory activity were seen when TRH injection was restricted to the lateral and third ventricles (forebrain). Furthermore, systemic administration of TRH (28 nmol) produced no significant respiratory effects. The active analogue, [3-Me-His2]-TRH (2.7 nmol) produced the same respiratory effects as TRH. The inactive analogue, TRH free acid (28-280 nmol), caused no significant change in respiratory activity. The data suggest that TRH interacts with a specific receptor in the hindbrain of the cat to affect respiration.

Respiratory depression produced by centrally administered taurine in the cat.

The effects of taurine (0.8-64.8 mumol) were studied on respiratory activity following intracisternal (cisterna magna) and intracerebroventricular (lateral ventricle) injections in cats anesthetized with alpha-chloralose. Respiratory activity was measured by using a Fleisch pneumotachograph and monitoring tracheal airflow. The flow signal was integrated to obtain tidal volume (VT) and respiratory rate (f) was obtained by counting the number of VT excursions over one minute. Inspiratory (TI), expiratory (TE) and total (TTOT) cycle durations were also determined during this time period. In addition, end-tidal CO2 was continuously monitored. Associated changes in arterial pressure (femoral artery cannula) and heart rate were also determined. After injections into the cisterna magna, taurine caused dose-related decreases in minute ventilation (VE). The maximal decrease in VE was from 495 +/- 59 to 64 +/- 14 ml/min (p less than 0.05), and was due to both decreases in VT (from 27 +/- 3 to 5 +/- 1 ml; p less than 0.05) and f (from 18 +/- 1 to 12 +/- 2 breaths/min; p less than 0.05). TE and TTOT were increased from 2.4 +/- 0.4 to 4.5 +/- 0.6 sec (p less than 0.05) and from 3.7 +/- 0.4 to 6.4 +/- 0.8 sec (p less than 0.05), respectively. Mean inspiratory flow (VT/TI), a measure of inspiratory drive, was decreased from 21 +/- 4 to 4 +/- 2 ml/sec (p less than 0.05). Apnea occurred in 5 of 6 animals after the 64.8 mumol dose. This respiratory depression occurred without any significant change in arterial pressure. After lateral ventricle injections, taurine also caused dose-related, but not as pronounced, decreases in respiratory activity. In addition, taurine caused significant decreases (p less than 0.05) in arterial pressure in doses that decreased VE. Taurine administered intravenously had no significant cardiorespiratory depressant effects. These data indicate that centrally administered taurine produces respiratory depression and, depending on the route of CNS administration, also produces hypotension.

Respiratory depression produced by glycine injected into the cisterna magna of cats.

Glycine (0.8-64.8 mumol) was administered into the cisterna magna of alpha-chloralose anesthetized cats to determine its effect on ventilation. Glycine caused a dose-related decrease in respiratory minute volume with the highest dose resulting in a decrease from 454 +/- 35 to 159 +/- 44 ml/min (p less than 0.05). This decrease was due primarily to a reduction in tidal volume which decreased from 31 +/- 2 to 12 +/- 2 ml (p less than 0.05). The two largest doses tested (21.6 and 64.8 mumol) also produced a decrease in respiratory rate from 14 +/- 1 to 11 +/- 1 and from 15 +/- 1 to 12 +/- 1 breaths/min (p less than 0.05), respectively. Apnea occurred in 2 of 7 animals given 64.8 mumol of glycine. No consistent dose-related changes in inspiratory and expiratory durations were observed. Intravenous administration of glycine (64.8 mumol) did not affect respiratory activity. These results indicate that glycine causes respiratory depression by an action in the central nervous system.