Gln

Figure 1. Effect of stimulation on the content and distribution of radiolabeled products of glutamate metabolism in axons and glia following 2 h incubation with [14C]-glutamate. Modified from data of Urazaev et al9. Means + 1 SEM shown in this figure were derived from the totality of samples analyzed, the statistical analysis was performed only on the 4 paired samples. Numbers in parentheses represent the percent of the sum of the radiolabeled compounds recovered in samples of axonal or glial cytoplasm averaged for each experimental pair. * Indicates statistical significance between stimulated and unstimulated nerve fibers at p<0.05.

depleted NAAG from the axon but not the glia, in contrast to that seen with nerve fibers bath incubated with radiolabel. In these same experiments it was also shown that nerve fibers incubated with radiolabeled NAAG and the NAAG peptidase inhibitors, P-NAAG or quisqualate, the superfusate concentration of glutamate was reduced by about 60% of the control suggesting that NAAG peptidase, with its hydrolytic site in the extracellular space, was present.

In a continuing series of experiments10, it was found that stimulation of nerve in the presence of radiolabeled NAAG dramatically increased the amount of NAAG, glutamate and glutamine in axons and their associated glia (Fig.2). These results and those described above suggest that the glial-associated NAAG peptidase was activated by stimulation to hydrolyze extracellular NAAG. The glutamate released can be taken up by glia and recycled into the NAAG, glutamate and glutamine pools of both axons and glia. In the absence of exogenous NAAG, endogenous NAAG released by stimulation is also hydrolyzed. The glutamate released can be detected as an increased NMDA receptor activation that potentiates the NAAG activated metabotropic glutamate receptor-initiated hyperpolarization response of glia (Fig.3). These results clearly point to the fact that axons and their associated glia utilize glutamate to synthesize NAAG and that NAAG is an axon-to-glia signaling agent that acts specifically on a glial group II metabotropic receptor to activate a number of physiological responses of the glia just now being identified9-11,19,20. These include, but not limited to, activation of signaling cascades that regulate glial membrane potential, extracellular K+ homeostasis and glial volume regulation and the activation of GCPII to terminate the NAAG signal. Glutamate, the

Figure 2: Effect of stimulation on accumulation and cellular distribution of radiolabeled metabolites of NAAG in medial giant nerve fibers. Panel A: The resting nerve cord was incubated with [3H]-NAAG for 30 min in the absence of 2-PMPA, a potent GCPII inhibitor. The number above each bar indicates the fraction of 14 axons and glia that had detectable radiolabel in NAAG, ASP, GLN and GLU. Mean radiolabel content is for all 14 fibers (samples in which there were no detectable radioactivity were given a value of zero). Panel B: Nerve fibers were incubated for 29 min incubation with [3H]-NAAG and then stimulated at 50 Hz during the 30th minute of incubation. The number of samples containing detectable amounts of radiolabeled NAAG, GLU and GLN increased dramatically for the 10-11 nerve fibers and the mean radiolabel content increased by a factor of 3 to 6. Data are presented as mean + 1 SEM. The axonal and glial contents of all radiolabeled substances with stimulation (panel B) were significantly greater than those of the unstimulated controls (panel A) at p < 0.001. Reprinted by permission from Urazaev et al10.

Figure 2: Effect of stimulation on accumulation and cellular distribution of radiolabeled metabolites of NAAG in medial giant nerve fibers. Panel A: The resting nerve cord was incubated with [3H]-NAAG for 30 min in the absence of 2-PMPA, a potent GCPII inhibitor. The number above each bar indicates the fraction of 14 axons and glia that had detectable radiolabel in NAAG, ASP, GLN and GLU. Mean radiolabel content is for all 14 fibers (samples in which there were no detectable radioactivity were given a value of zero). Panel B: Nerve fibers were incubated for 29 min incubation with [3H]-NAAG and then stimulated at 50 Hz during the 30th minute of incubation. The number of samples containing detectable amounts of radiolabeled NAAG, GLU and GLN increased dramatically for the 10-11 nerve fibers and the mean radiolabel content increased by a factor of 3 to 6. Data are presented as mean + 1 SEM. The axonal and glial contents of all radiolabeled substances with stimulation (panel B) were significantly greater than those of the unstimulated controls (panel A) at p < 0.001. Reprinted by permission from Urazaev et al10.

hydrolysis product of NAAG, primarily acts on glial NMDA receptors participating in a number of cellular activities including glial membrane potential regulation, increased intracellular Ca2+ and NO production.

Figure 3: The effect of 2PMPA and MK 801 on the amplitude of the stimulation-induced glial cell hyperpolarization. The data of this figure demonstrates that the amplitude of glial cell hyperpolarization increased proportionally with increasing stimulation frequency between 25 and 100Hz. The GCP II inhibitor, 2-PMPA (0.1 ^M), or the NMDA antagonist, MK801 (10 ^M) blocked a portion of the hyperpolarization to nearly the same extent at each stimulation frequency. Data are presented as mean + SEM. Asterisks indicate a statistically significant difference from the control at p < 0.05 (n=16-20). Reprinted by permission from Urazaev et al .10

Figure 3: The effect of 2PMPA and MK 801 on the amplitude of the stimulation-induced glial cell hyperpolarization. The data of this figure demonstrates that the amplitude of glial cell hyperpolarization increased proportionally with increasing stimulation frequency between 25 and 100Hz. The GCP II inhibitor, 2-PMPA (0.1 ^M), or the NMDA antagonist, MK801 (10 ^M) blocked a portion of the hyperpolarization to nearly the same extent at each stimulation frequency. Data are presented as mean + SEM. Asterisks indicate a statistically significant difference from the control at p < 0.05 (n=16-20). Reprinted by permission from Urazaev et al .10

3.2 NAAG Synthesis in Axoplasm Isolated from the Medial Giant Axon of the Crayfish

Synthesis of NAAG and NAA (Figs. 4, 5) was examined in axoplasm isolated from the axon by microcannulae as described in the METHODS. Radiolabeled glutamate, used as the substrate for NAAG synthesis, was added to isolated axoplasm in distilled H2O or in an incubation medium (IM) containing in mM; .25 NAA; 0.1 cAMP; 0.5 ATP; 50 KCL; 10 MgCl2; 30 Tri.HCl at pH 7.03. The volume of IM or glutamate in water as a fraction of the axoplasm volume varied from 1% to 25% depending on the protocol. Incubation of the axoplasm with the IM was 2-3 hours at room temperature. Following incubation, the axoplasm droplets were diluted in 100^ of water containing non-labeled GLU (100nM), NAA (100nM) and NAAG (50nM) to facilitate identification of NAA and NAAG peaks with A TSK-GEL SAX coulmn HPLC system. The individual peaks were collected and analyzed for radioactivity.

In the experiments illustrated in Fig. 4, radiolabeled glutamate was added only to track synthesis and represented approximately 10-9 M. This amount was insignificant with respect to the endogenous glutamate in native isolated axoplasm of approximately (10-2 M). When radiolabeled glutamate in water was added to axoplasm, in the absence of cofactors or other substrates, little NAAG was synthesized (filled triangles) even up to a volume fraction of 0.25. On the other hand, with incubation medium (IM) (filled squares) there was a steep slope of increasing NAAG synthesis to approximately 100 pmoles/^.hr at volume fraction of 0.17. In a direct comparison between glutamate in water and IM solutions at a volume fraction of 0.1, synthesis of NAAG was 6.4+3.7 pmole/^.hour (n=4) compared to 56.8+4.2 pmole/^.hour, respectively. These results suggest that NAA and/or one or more of the cofactors in IM are necessary for the efficient synthesis of NAAG.

Volume IM:Volume(axoplasm)

Figure 4. The effects of increasing radiolabeled glutamate, NAA and cofactors on NAAG synthesis in isolated axoplasm.

Volume IM:Volume(axoplasm)

Figure 4. The effects of increasing radiolabeled glutamate, NAA and cofactors on NAAG synthesis in isolated axoplasm.

For the experiments illustrated in Fig. 4 our initial assumption was that there would be sufficient substrate in native axoplasm to support synthesis using [14C]-glutamate in water as the only additive. As seen that is clearly not the case. Basing the protocol on the above described experiments, NAA was the first to be examined as a substrate for NAAG synthesis. These experiments were performed with a constant volume fraction of IM solution, radiolabeled glutamate to track synthesis and varying amounts of NAA. The results are shown in Fig. 5.

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