NAAG peptidase inhibitors block cognitive deficit induced by MK-801 and motor activation induced by d-amphetamine in animal models of schizophrenia.

The most widely validated animal models of the positive, negative and cognitive symptoms of schizophrenia involve administration of d-amphetamine or the open channel NMDA receptor blockers, dizocilpine (MK-801), phencyclidine (PCP) and ketamine. The drug ZJ43 potently inhibits glutamate carboxypeptidase II (GCPII), an enzyme that inactivates the peptide transmitter N-acetylaspartylglutamate (NAAG) and reduces positive and negative behaviors induced by PCP in several of these models. NAAG is an agonist at the metabotropic glutamate receptor 3 (mGluR3). Polymorphisms in this receptor have been associated with expression of schizophrenia. This study aimed to determine whether two different NAAG peptidase inhibitors are effective in dopamine models, whether their efficacy was eliminated in GCPII knockout mice and whether the efficacy of these inhibitors extended to MK-801-induced cognitive deficits as assessed using the novel object recognition test. ZJ43 blocked motor activation when given before or after d-amphetamine treatment. (R,S)-2-phosphono-methylpentanedioic acid (2-PMPA), another potent NAAG peptidase inhibitor, also reduced motor activation induced by PCP or d-amphetamine. 2-PMPA was not effective in GCPII knockout mice. ZJ43 and 2-PMPA also blocked MK-801-induced deficits in novel object recognition when given before, but not after, the acquisition trial. The group II mGluR antagonist LY341495 blocked the effects of NAAG peptidase inhibition in these studies. 2-PMPA was more potent than ZJ43 in a test of NAAG peptidase inhibition in vivo. By bridging the dopamine and glutamate theories of schizophrenia with two structurally different NAAG peptidase inhibitors and demonstrating their efficacy in blocking MK-801-induced memory deficits, these data advance the concept that NAAG peptidase inhibition represents a potentially novel antipsychotic therapy.

NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance.

The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) where it acts to decrease cAMP levels. Rat cortical interneurons express both NAAG and glutamic acid decarboxylase, as well as mGluR3 mRNA. In the presence of ionotropic glutamate receptor antagonists, both NAAG and the group II metabotropic glutamate receptor agonist, DCG-IV, reduced the calcium-dependent, KCl-induced [(3)H]-GABA release from rat cortical neurons by 35%. This release process was unaffected by tetrodotoxin. The group II antagonist, ethyl glutamate, reversed the effects of DCG-IV and NAAG. The mGluR3-selective antagonist, beta-N-acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl-stimulated [(3)H]-GABA-release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H-89, decreased [(3)H]-GABA release while NAAG produced no additional inhibition in the presence of H-89. In contrast, the protein kinase C inhibitor, Ro 31--8220, had no effect on KCl-stimulated release, nor did it affect the inhibition of release produced by NAAG. The L-type calcium channel blocker, nifedipine, also inhibited the release of [(3)H]-GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl-stimulated [(3)H]-GABA release by NAAG is mediated via presynaptic mGluR3 on GABAergic cortical neurons and that this effect is obtained by decreasing cAMP with a consequent decrease in protein kinase A activity and L-type calcium channel conductance.

N-Acetylaspartylglutamate: the most abundant peptide neurotransmitter in the mammalian central nervous system.

In the progress of science, as in life, timing is important. The acidic dipeptide, N-acetylaspartylglutamate (NAAG), was discovered in the mammalian nervous system in 1965, but initially was not considered to be a neurotransmitter candidate. In the mid-1980s, a few laboratories revisited the question of NAAG's role in the nervous system and pursued hypotheses regarding its function that ranged from a precursor for the transmitter pool of glutamate to a direct role as a peptide transmitter. Since that time, NAAG has been tested against nearly all of the established criteria for identification of a neurotransmitter. It successfully meets each of these tests, including a concentrated presence in neurons and synaptic vesicles, release from axon endings in a calcium-dependent manner following initiation of action potentials, and extracellular hydrolysis by membrane-bound peptidase activity. NAAG is the most prevalent and widely distributed neuropeptide in the mammalian nervous system. NAAG activates NMDA receptors with a low potency that may vary among receptor subtypes, and it is a highly selective agonist at the type 3 metabotropic glutamate receptor (mGluR3). Acting through this receptor, NAAG reduces cyclic AMP levels, decreases voltage-dependent calcium conductance, suppresses excitotoxicity, influences long-term potentiation and depression, regulates GABA(A) receptor subunit expression, and inhibits synaptic release of GABA from cortical neurons. Cloning of peptidase activities against NAAG provides opportunities to study the cellular and molecular mechanisms by which synaptic NAAG peptidase activity is controlled. Given the codistribution of this peptide with a spectrum of traditional transmitters and its ability to activate mGluR3, we speculate that one role for NAAG following synaptic release is the activation of metabotropic autoreceptors that inhibit subsequent transmitter release. A second role is the production of extracellular glutamate following NAAG hydrolysis.

Molecular cloning of a peptidase against N-acetylaspartylglutamate from a rat hippocampal cDNA library.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate-specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752-amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by beta-NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5' end, the central region, and the 3' end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5' end of the hippocampal cDNA.

Regional expression and chromosomal localization of the delta opiate receptor gene.

The delta opiate receptor gene has been cloned from the mouse neuroblastoma-rat glioma hybrid cell NG108-15. The clone that we isolated is apparently identical to that reported by Evans et al. [Evans, C. J., Keith, D. E., Jr., Morrison, H., Magendzo, K. & Edwards, R. H. (1992) Science 258, 1952-1955] and essentially identical with that of Kieffer et al. [Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C. & Hirth, C. G. (1992) Proc. Natl. Acad. Sci. USA 89, 12048-12052]. We have found full-length transcripts of the gene in mouse brain but in no other tissues examined. Within the brain the gene is expressed at low levels in many regions but transcripts are found in particularly large amounts in the anterior pituitary and pineal glands. Since these tissues are located outside the blood-brain barrier, opioid peptides easily can reach receptors in these areas from the blood. The gene, which is present as a single copy, has been mapped to the distal region of mouse Chromosome 4.

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.

Regulation of the erythrocyte Ca2(+)-ATPase by mutant calmodulins with positively charged amino acid substitutions.

Four mutant calmodulins with site-specific charge alterations have been used to activate the human erythrocyte Ca2(+)-ATPase. These charge alterations were accomplished either by insertion of new Lys residues or by substitution of Lys residues for Glu in two of the seven calmodulin alpha-helices. Two enzyme preparations, purified monomeric Ca2(+)-ATPase and erythrocyte ghost membranes, were used with comparable results. At 100 nM Ca2+, the Ca2(+)-ATPase activity was lowered significantly by charge reversal from negative to positive in both the central alpha-helix and the carboxy-terminal domain. While all mutant calmodulins with charge reversal ultimately stimulated the Ca2(+)-ATPase activity to the same extent, the concentration of mutant calmodulin required for half-maximal activation was from 36-fold (central alpha-helix) to 126-fold higher (alpha-helix in the carboxy-terminal domain) than that of the control calmodulin. There was also a significant difference in the stimulation of Ca2(+)-ATPase activity by the different mutant calmodulins as a function of Ca2+ concentration, being most pronounced at submicromolar Ca2+ concentrations where enzyme activation by calmodulin appears to be a physiologically relevant mechanism. In contrast to the mutant calmodulins with charge reversal, mutant calmodulins in which two positive charges were added in the central alpha-helix activated the Ca2(+)-ATPase in a way undistinguishable from the control calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of calmodulin on erythrocyte Ca2(+)-ATPase activation and oligomerization.

The study was performed on the purified human erythrocyte Ca2(+)-ATPase to test whether or not calmodulin promotes enzyme oligomerization. Two physiologically significant modes of activation of this enzyme were considered, by calmodulin binding to monomeric enzyme and by enzyme oligomerization [Kosk-Kosicka & Bzdega (1988) J. Biol. Chem. 263, 18184]; it was not clear whether the two modes were interdependent or operated independently. Fluorescence resonance energy transfer (FRET) between separately labeled Ca2(+)-ATPase molecules was used to monitor oligomerization. No change in energy transfer efficiency was observed upon subsequent addition of calmodulin at different enzyme concentrations. Lack of decrease in the enzyme concentration at which the half-maximal oligomerization occurred indicated that calmodulin did not facilitate oligomerization. The calmodulin inhibitor compound 48/80 had no effect on either the Ca2(+)-ATPase activity of oligomers or the extent of oligomerization measured by FRET while it drastically decreased the calmodulin-stimulated activity of the monomeric Ca2(+)-ATPase. The findings demonstrate that calmodulin is not involved in the oligomerization-induced activation pathway; it neither promotes oligomerization nor stimulates the Ca2(+)-ATPase activity of oligomers. We have demonstrated that calmodulin added before mixing donor- and acceptor-labeled enzyme populations prevented the occurrence of energy transfer. This inhibition of the formation of mixed donor-acceptor enzyme oligomers by calmodulin was dose dependent. Also, the reversal of the inhibition by compound 48/80 proceeded in a dose-dependent manner. Further, calmodulin prevented the apparent decrease of energy transfer efficiency that resulted from dilution of mixed donor-acceptor enzyme oligomers with unlabeled enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states.

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Ca2(+)-ATPase: activation by enzyme oligomerization versus by calmodulin.

The subject of our studies is the mechanism of activation of the erythrocyte Ca2(+)-ATPase. Using purified, detergent solubilized enzyme it was found that equivalent maximal Ca2(+)-ATPase activity is obtained either upon addition of calmodulin or upon increase of enzyme concentration. Three independent methods, including Ca2(+)-ATPase activity, polarization of the enzyme modified with an external fluorescent probe, and efficiency of fluorescence resonance energy transfer between enzyme molecules have established that the concentration dependent activation is due to enzyme oligomerization. The oligomers bind calmodulin with a lower stoichiometry (0.5 mol calmodulin/mol Ca2(+)-ATPase), higher Ca2+ affinity (KCa = pCa 7.4), and higher cooperativity for Ca2+ (nH = 2.6) than the monomeric form (stoichiometry = 1 mol calmodulin/mol Ca2(+)-ATPase, KCa = pCa 7.0, nH = 1.1). The Ca2+ dependence of calmodulin binding and activation of monomers indicates that calmodulin binds before the Ca2(+)-ATPase activity is exhibited, demonstrating that the activation of this enzyme form is totally dependent on calmodulin. In contrast, oligomers reveal very similar Ca2+ dependence for calmodulin binding and for Ca2(+)-ATPase activity as well as for Ca2+ binding (assessed by tryptophan fluorescence), and for the oligomerization process (assessed by fluorescence energy transfer). The calmodulin antagonist drug 48/80 inhibits the calmodulin dependent activity of the monomers (I50 = 1.4 micrograms/ml) but has no effect on the activity of oligomers, confirming that calmodulin plays no role in the activation of the oligomeric enzyme. Our studies indicate that the erythrocyte Ca2(+)-ATPase can be activated by its high affinity, Ca2+ dependent binding of calmodulin or by a Ca2+ dependent oligomerization process which may involve calmodulin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.

Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5'-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed a sigmoid dependence on enzyme concentration and was half-maximal at 10-12 nM enzyme; this dependence on enzyme concentration closely resembled previously demonstrated dependence of Ca2+-ATPase activity and polarization of the fluorescein 5'-isothiocyanate enzyme (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189). Thus, the three independent methods establish that enzyme concentration-dependent oligomerization is a mechanism of activation of the erythrocyte Ca2+-ATPase. Further energy transfer studies demonstrated that enzyme oligomerization required calcium. This calcium dependence was characterized by high affinity (half-maximal energy transfer at pCa 7.15) and cooperativity (Hill coefficient of 2.36), being very similar in both respects to the Ca2+ dependence of the Ca2+-ATPase activity. The data indicated that the oligomerization process produced a highly cooperative, Ca2+-regulated activation of the enzyme at physiologically relevant Ca2+ concentrations. These studies show that the Ca2+-ATPase can be fully activated by a Ca2+-dependent oligomerization mechanism, which is independent of the previously described activation by calmodulin. We propose two pathways for the activation of the Ca2+-ATPase, taking into account the interdependencies between the Ca2+, calmodulin, and enzyme concentrations.

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin.

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.