Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

N-acetylaspartylglutamate (NAAG) is the probable mediator of axon-to-glia signaling in the crayfish medial giant nerve fiber.

Glial cell hyperpolarization previously has been reported to be induced by high frequency stimulation or glutamate. We now report that it also is produced by the glutamate-containing dipeptide N-acetylaspartylglutamate (NAAG), by its non-hydrolyzable analog beta-NAAG, and by NAAG in the presence of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a potent inhibitor of the NAAG degradative enzyme glutamate carboxypeptidase II. The results indicate that NAAG mimics the effect of nerve fiber stimulation on the glia. Although glutamate has a similar effect, the other presumed product of NAAG hydrolysis, N-acetylaspartate, is without effect on glial cell membrane potential, as is aspartylglutamate (in the presence of 2-PMPA). The hyperpolarization induced by stimulation, glutamate, NAAG, beta-NAAG, or NAAG plus 2-PMPA is completely blocked by the Group II metabotropic glutamate receptor antagonist (S)-alpha-ethylglutamate but is not altered by antagonists of Group I or III metabotropic glutamate receptors. The N-methyl-D-aspartate receptor antagonist MK801 reduces but does not eliminate the hyperpolarization generated by glutamate, NAAG or stimulation. These results, in combination with those of the preceding paper, are consistent with the premise that NAAG could be the primary axon-to-glia signaling agent. When the unstimulated nerve fiber is treated with cysteate, a glutamate reuptake blocker, there is a small hyperpolarization of the glial cell that can be substantially reduced by pretreatment with 2-PMPA before addition of cysteate. A similar effect of cysteate is seen during a 50 Hz/5 s stimulation. From these results we suggest that glutamate derived from NAAG hydrolysis appears in the periaxonal space under the conditions of these experiments and may contribute to the glial hyperpolarization.

Uptake and metabolism of glutamate at non-synaptic regions of crayfish central nerve fibers: implications for axon-glia signaling.

In crayfish and squid giant nerve fibers, glutamate appears to be an axon-glia signaling agent. We have investigated glutamate transport and metabolism by crayfish central nerve fibers in order to identify possible mechanisms by which glutamate could subserve this non-synaptic signaling function. Accumulation of radiolabeled L-glutamate by desheathed cephalothoracic nerve bundles was temperature and Na(+) dependent, linear with time for at least 8h and saturable at about 0.5-1mM L-glutamate. Most accumulated radiotracer was associated with the periaxonal glial sheath and remained as glutamate. Compounds known to block glutamate transport in invertebrate peripheral nerves or mammalian brain slices or cell cultures were also effective on crayfish central nerve fibers. Tissue radiotracer levels were only 3% of control levels when 1mM p-chloromercuriphenylsulfonate was present, and 13%, 20%, 26%, 38% and 42% of control levels, respectively, when L-cysteate, L-cysteine sulfinate, L-aspartate, D-aspartate or DL-threo-beta-hydroxyaspartate was present. L-Glutamine, GABA, N-methyl-DL-aspartate, alpha-aminoadipate and D-glutamate were without inhibitory effect on tissue tracer accumulation. Radiolabeled D-aspartate was an equivalent non-metabolized substitute for radiolabeled L-glutamate. D-Aspartate, p-chloromercuriphenylsulfonate and GABA had comparable effects on isolated medial giant nerve fibers.These studies indicate that L-glutamate is taken up primarily by the periaxonal glia of crayfish central nerve fibers by a low-affinity, saturable, Na(+)-dependent transport system and is retained by the fibers primarily in that form. Our results suggest that the glia are not only the target of the glutamate signal released from non-synaptic regions of the crayfish medial giant axon during high-frequency stimulation, but that they are also the primary site of its inactivation.

Properties of glutaminase of crayfish CNS: implications for axon-glia signaling.

Glutaminase of crayfish axons is believed to participate in recycling of axon-glia signaling agent(s). We measured the activity and properties of glutaminase in crude homogenates of crayfish CNS, using ion exchange chromatography to separate radiolabeled product from substrate. Crayfish glutaminase activity is cytoplasmic and/or weakly bound to membranes and dependent on time, tissue protein, and glutamine concentration. It resembles the kidney-type phosphate-activated glutaminase of mammals in being stimulated by inorganic phosphate and alkaline pH and inhibited by the product glutamate and by the glutamine analog 6-diazo-5-oxo-L-norleucine. During incubation of crayfish CNS fibers in Na(+)-free saline containing radiolabeled glutamine, there is an increased formation of radiolabeled glutamate in axoplasm that is temporally associated with an increase in axonal pH from about 7.1 to about 8.0. Both the formation of glutamate and the change in pH are reduced by 6-diazo-5-oxo-L-norleucine. Our results suggest that crayfish glutaminase activity is regulated by cellular changes in pH and glutamate concentration. Such changes could impact availability of the axon-glia signaling agents glutamate and N-acetylaspartylglutamate.

Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon-glia signaling.

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.

Mechanisms for clearance of released N-acetylaspartylglutamate in crayfish nerve fibers: implications for axon-glia signaling.

Crayfish nerve fibers incubated with radiolabeled glutamate or glutamine accumulate these substrates and synthesize radioactive N-acetylaspartylglutamate (NAAG). Upon stimulation of the medial giant nerve fiber, NAAG is the primary radioactive metabolite released. Since NAAG activates a glial hyperpolarization comparable to that initiated by glutamate or axonal stimulation through the same receptor, we have proposed that it is the likely mediator of interactions between the medial giant axon and its periaxonal glia. This manuscript reports investigations of possible mechanisms for termination of NAAG-signaling activity. N-acetylaspartyl-[(3)H]glutamate was not accumulated from the bath saline by unstimulated crayfish giant axons or their associated glia during a 30-min incubation. Stimulation of the central nerve cord at 50 Hz during the last minute of the incubation dramatically increased the levels of radiolabeled glutamate, NAAG, and glutamine in the medial giant axon and its associated glia. These results indicate that stimulation-sensitive peptide hydrolysis and metabolic recycling of the radiolabeled glutamate occurred. There was a beta-NAAG-, quisqualate- and 2-(phosphonomethyl)-pentanedioic acid-inhibitable glutamate carboxypeptidase II activity in the membrane fraction of central nerve fibers, but not in axonal or glial cytoplasmic fractions. Inactivation of this enzyme by 2-(phosphonomethyl)-pentanedioic acid or inhibition of N-methyl-D-aspartate (NMDA) receptors by MK801 reduced the glial hyperpolarization activated by high-frequency stimulation. These results indicate that axon-to-glia signaling is terminated by NAAG hydrolysis and that the glutamate formed contributes to the glial electrical response in part via activation of NMDA receptors. Both NAAG release and an increase in glutamate carboxypeptidase II activity appear to be induced by nerve stimulation.

Axon-glia exchange of macromolecules.

The periaxonal and perineurial glia of crayfish and squid are strategically situated to regulate the neuronal microenvironment. Diverse molecules rapidly traverse the periaxonal sheath and a fraction of them enters the axons from glia or the glia from axons. The significance of these intercellular exchanges has not been tested directly. However, recent reports suggest that stress proteins, which probably are synthesized by both types of glia and transferred to axons, may be essential components by which the glia directly and indirectly assist neurons in tolerating ambient stress.

Glutamine uptake and metabolism to N-acetylaspartylglutamate (NAAG) by crayfish axons and glia.

We have proposed that N-acetylaspartylglutamate (NAAG) or its hydrolytic product glutamate, is a chemical signaling agent between axons and periaxonal glia at non-synaptic sites in crayfish nerves, and that glutamine is a probable precursor for replenishing the releasable pool of NAAG. We report here, that crayfish central nerve fibers synthesize NAAG from exogenous glutamine. Cellular accumulation of radiolabel during in vitro incubation of desheathed cephalothoracic nerve bundles with [3H]glutamine was 74% Na(+)-independent. The Na(+)-independent transport was temperature-sensitive, linear with time for at least 4 h, saturable between 2.5 and 10 mM L-glutamine, and blocked by neutral amino acids and analogs that inhibit mammalian glutamine transport. Radiolabeled glutamine was taken up and metabolized by both axons and glia to glutamate and NAAG, and a significant fraction of these products effluxed from the cells. Both the metabolism and release of radiolabeled glutamine was influenced by extracellular Na(+). The uptake and conversion of glutamine to glutamate and NAAG by axons provides a possible mechanism for recycling and formation of the axon-to-glia signaling agent(s).

Cloning and sequence analysis of a cDNA for an inducible 70 kDa heat shock protein (Hsp70) of the American oyster (Crassostrea virginica).

We have been investigating 70 kDa heat shock proteins (Hsp70s) as potential molecular markers for improved breeding and stress management to revitalize stocks of the American oyster, C. virginica. From a C. virginica visceral mass library, a 2.2 kb full-length cDNA was isolated that included a 634 amino acid open reading frame possessing approximately 80% sequence identity with inducible and constitutive Hsp70s of a broad array of animal species. Northern blotting indicated that the cloned cDNA preferentially recognized an mRNA of about 2 kb that was virtually absent from visceral mass under basal conditions but greatly increased after in vivo heat shock of American and Pacific oysters, suggesting that the cDNA codes for an inducible Hsp70.

Inhibition of crayfish glutamic acid decarboxylase by structural analogs of the substrate and product.

Crayfish glutamic acid decarboxylase (GAD) is inhibited by some aliphatic carboxylic acid analogs of glutamate and gamma-amino-n-butyric acid (GABA). Variations in the length of the carbon skeleton, substitution of a keto for a methylene group, replacement of the carboxyl group or attachment of a bulky basic moiety to the amino terminus of GABA all lead to a drastic reduction in its inhibitory activity. Substitution of a methyl group for the amino group of GABA is a permissible alteration which does not reduce the inhibitory potency. Some structural analogs of glutamate are inhibitory also, particularly if they possess a comparable carbon skeleton and a keto group in the alpha position or a sulfhydryl group. Most of the sulfhydryl analogs are significantly more potent as inhibitors than the corresponding compounds in which the SH group is replaced by an H atom.

Stress protein synthesis and accumulation after traumatic injury of crayfish CNS.

By several days after a crush injury of crayfish CNS, the wound site heals. Changes in protein synthesis and accumulation occur at the lesion site and nearby. During the first few hours, synthesis of 35, 70, 90, and 150 kDa proteins is induced in the injured tissue. By one day, the relative amounts of 70-90 kDa proteins increase dramatically, particularly at the crush site and adjacent to it. The 70 kDa proteins, which are related to mammalian stress proteins (SPs), remain elevated for at least one month in the traumatized region or nearby. The crushed tissue contains an SP70 isoform not present in its uncrushed counterpart. These biochemical changes may reflect the cellular changes that accompany wound healing and/or a cellular stress response to compensate for the lesion. Since similar adaptations occur in the mammalian CNS, they may represent a phylogenetically conserved attempt to retard or repair CNS tissue deterioration.

Long-term persistence of GAD activity in injured crayfish CNS tissue.

Crayfish CNS fibers were isolated in vivo from their cell bodies, from cellular connections in the CNS, and from peripheral sensory and effector cells. The glutamic acid decarboxylase (GAD) activity of the experimental tissues was about half of that of the sham-operated and unoperated control tissues by two weeks after surgery and remained at about that level during the ensuing six weeks. During that time, there was no significant behavioral, electrophysiological, or histological evidence of regeneration of nerve fibers across the lesion sites. The crush-isolated connectives possessed many intact axon profiles and non-neuronal cell nuclei. The long-term persistence of GAD activity in the injured CNS tissue may reflect the involvement of glial cells in maintaining neurotransmitter levels.

Inhibitors of crayfish glutamic acid decarboxylase.

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5'-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN- and Cl-), and some cations (e.g. Zn+2). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.

Assay and properties of glutamic acid decarboxylase in homogenates of crayfish nervous tissue.

The activity of glutamic acid decarboxylase (GAD) was measured in homogenates of crayfish nervous tissue. Radioactive GABA and CO2 were formed from radioactive glutamic acid in approximately equimolar amounts. Product formation was linear for 9.5 hr at 11-32 degrees C with about 1-30 micrograms homogenate protein. Enzyme activity remained high at pH 7-10 but declined steeply above pH 10.5 and below pH 7. Enzyme activity was stimulated by pyridoxal phosphate, 2-mercaptoethanol, and potassium phosphate; at higher than optimal concentrations of each the activity was reduced. Sodium phosphate altered the stimulatory effect of potassium phosphate. Crayfish GAD behaves like a typical neural GAD but is distinguishable biochemically from GAD of other species.

Axon-glia transfer of a protein and a carbohydrate.

We have investigated the transfer of a fluorescent protein, the fluorescein isothiocyanate derivative of bovine serum albumin (FITC-BSA), and a fluorescent carbohydrate, FITC-dextran, from the crayfish medial giant axon (MGA) to the periaxonal glial cells. The dialyzed tracer was injected into one of the two MGAs, and, after a transfer period of 15-60 min, the tissue was fixed for histological examination of fluorescence distribution. With each tracer, the periaxonal sheath of the injected MGA was specifically labeled. Similar results were obtained with several different fixatives. During the transfer period, there was no appreciable change in the resting potential or conducted action potential of the MGA or in the resting potentials of the adaxonal glial cells. Polyacrylamide gel electrophoresis indicated that the axoplasmic and sheath fluorescence was produced by the intact tracers. These results suggest that "foreign" macromolecules can be exchanged from crayfish axons to glia under physiological conditions. Such transfers may indicate a substantial intercellular traffic of molecules or a means whereby neurons can eliminate waste materials.

Glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling.

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.

Stress protein synthesis by crayfish CNS tissue in vitro.

Some crustacean axons remain functional for months after injury. This unusual property may require stress proteins synthesized by those neurons or provided to them by glial cells. To begin to explore this hypothesis, we examined the conditions that stimulated stress protein synthesis by crayfish CNS tissue in vitro. Incubation for 1-15 h with arsenite or at temperatures about 15 degrees C higher than the acclimation temperature of 20 degrees C induced transient expression of several stress proteins. The heat stress response was blocked by Actinomycin D, suggesting that synthesis of new mRNA was required. In addition, the major crayfish 66 kD stress protein and its mRNA had sequence identities with the 70 kD stress proteins of mammals. Since the crayfish stress response has much in common with that of other organisms, the unique advantages of the crayfish nervous system can be used to study the impact of stress proteins on glial and neuronal function.

[Synthesis and release of N-acetylaspartyl glutamate (NAAG) in medial giant axons in crayfish]. / Sintez i osvobozhdenie N-acetylaspartylglutamate (NAAG) v sredinnykh gigantskikh aksonakh raka.

Studies of crayfish Medial Giant nerve Fiber suggested that glutamate (GLU) released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. This investigation sought to elucidate the mechanism of GLU appearance extracellularly following axon stimulation. Axoplasm and periaxonal glial sheath from nerve fibers incubated with radiolabelled L-GLU contained radiolabeled GLU, glutamine (GLN), GABA, aspartate (ASP), and NAAG. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [14C]-GLU by bath application or loaded with [14C]-GLU, [3H]-D-ASP, or [3H]-NAAG by axonal injection. However, radioactivity distribution among GLU and its metabolic products in the superfusate was changed, with NAAG accounting for the largest fraction. In axons incubated with radiolabeled GLU, the stimulated increase in radioactive NAAG in the superfusate coincided with the virtual clearance of radioactive NAAG from the axon. The increase in [3H]-GLU in the superfusion solution that was seen upon stimulation of nerve bathloaded with [3H]-NAAG was reduced when beta-NAAG, a competitive NAALADase inhibitor, was present. Together, these results suggest that some GLU is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released and converted to GLU by NAALADase. A quisqualate-, beta-NAAG-sensitive NAALADase activity was detected in nerve cord homogenates. Stimulation or NAAG administration in the presence of NAALADase inhibitor caused a transient hyperpolarization of the periaxonal glia comparable to that produced by L-GLU. The results implicate N-acetylaspartylglutamate (NAAG) and GLU as potential mediators. of the axon-glia interactions.