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The action of nitric oxide (NO) synthesized by NO synthases (NOS) is spatially restricted. Hence, the intracellular location of NOS might play an important role for the functional interactions of NO with its target molecules. In the skeletal muscle the neuronal NOS (nNOS) is considered to be the predominant isoform expressed as a muscle specific elongated splice variant. There are only a few and highly discrepant reports of the subcellular distribution of nNOS, which prompted us to re-examine the distribution of nNOS in the skeletal muscle of rat and mouse applying immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry. Light microscopically, the sarcolemma, areas beneath the sarcolemma, areas around the nuclei, and the cross striation were labeled by antibodies and by the NADPH-d reaction as well. Ultrastructurally, nNOS visualized immunocytochemically or by the histochemical BSPT-reaction, was associated discretely with extrajunctional portions of the sarcolemma. Both reaction products were additionally observed in the vicinity of endoplasmic reticulum and mitochondria, or associated with their outer membranes. In the neuromuscular junction (NMJ)-region NOS was localized to the cytoplasm of nerve terminals and terminal Schwann cells. In contrast to the commonly accepted assumption, the enzyme was found in association with the presynaptic, and not with the postsynaptic membrane. Cytosolic NADPH-d was exhibited especially between mitochondria accumulated in the postsynaptic region of the NMJ. Surprisingly, in nNOS −/− -mice the skeletal muscle showed patterns of significant nNOS-immunoreactivity and NADPH-d activity possibly due to alternative nNOS-splice isoforms, which might be up-regulated to compensate for decreased NO formation.

To examine the cellular distribution of radical scavenging enzymes in glia, in comparison to that in neurons and their behaviour during excitotoxically induced neurodegenerative processes, protein levels and the cellular localization of cytosolic and mitochondrial superoxide dismutase (Cu/Zn- and Mn-SOD) were investigated in the rat brain undergoing quinolinic acid (Quin)-induced neurodegeneration. Evidence for the specificity of the applied antibodies to detect immunocytochemically these SOD isoforms was obtained from electron microscopy and Western blotting. In control striatum Mn-SOD was clearly confined to neurons, whereas Cu/Zn-SOD was found, rather delicately, only in astrocytes. Microglia failed to stain with antibodies to both SOD isoforms. Quin application resulted in an initial formation of oxygen and nitrogen radicals as determined by the decline in the ratio of ascorbic to dehydroascorbic acid and by increased levels of nitrated proteins, an indicator for elevated peroxynitrite formation. Morphologically, massive neuronal damage was seen in parallel. Astroglia remained intact but showed initially decreased glutamine synthetase activities. The levels of Mn-SOD protein increased 2-fold 24 h after Quin injection (Western blotting) and declined only slowly over the time period considered (10 days). Cu/Zn-SOD levels increased only 1.3-fold. Immunocytochemical studies revealed that the increase in Mn-SOD is confined to neurons, whereas that of Cu/Zn-SOD was observed only in astroglial cells. Quiescent microglial cells were, as a rule, free of immunocytochemically detectable SOD, whereas in activated microglia a few Mn-SOD immunolabeled mitochondria occurred. Our results suggest a differential protective response in the Quin lesioned striatum in that Mn-SOD is upregulated in neurons and Cu/Zn-SOD in astroglia. Both SOD-isoforms are assumed to be induced to prevent oxidative and nitric oxide/peroxynitrite-mediated damage. In the border zone of the lesion core this strategy may contribute to resist the noxious stimulus.

Alterations in function and specific cellular location of cytoskeletal elements are characterized by changes in their phosphorylation state. On this background we studied immunocytochemically the distribution pattern of neurofilament (NF) in its phosphorylated (P-NF) and nonphosphorylated (NP-NF) form and of microtubule-associated protein-2 (MAP-2) in the rat and mouse brain. Neurons that are strongly positive for neuronal nitric oxide synthase (nNOS)-immunoreactivity (IR) showed, interestingly, neither P-NF- nor MAP-2-IR. In contrast, nNOS-negative neuronal cell elements exhibited an intense IR and specific location for both antigens throughout the brain. As a model we chose the dorsolateral tegmental nucleus (LDT) of knockout (nNOS(-/-)) mice in which the main splice isoform nNOSalpha is lacking, but a low nNOS-activity persists, apparently due to the splice isoforms nNOSbeta and gamma. The principal neurons of such nNOS(-/-)-mice, which are equivalent to the nNOS-containing neurons in the LDT of wild-type mice exhibited a decreased nitrotyrosine-IR and an increased phosphotyrosine-IR if compared to those of wild-type mice. The same neurons failed to show NF-IR and MAP-2-IR, though. When the residual nNOS activity in nNOS(-/-)-mice was inhibited by treatment with N-omega-nitro-L-arginine methyl ester (L-NAME) the principal neurons displayed a moderate MAP-2 and NF-staining. NO and NO-derived oxygen species are suggested to modulate the balance between the activities of kinases and phosphatases, thus changing phosphorylation levels for NF, MAP-2, and, possibly, other proteins in neurons and adjacent cell elements.

In homogenate supernatants of hippocampal formation and cerebellum of the rat, gamma-glutamyl transpeptidase (gamma-GTP) activities increased about 6 times from postnatal day 6 (0.178 +/- 0.02 and 0.187 +/- 0.007 U/g wet wt., respectively) to day 100. In dorsal root ganglia (0.183 +/- 0.003 U/g at day 6) and superior cervical ganglia (0.188 +/- 0.019 U/g at day 6) in which apparently amino acidergic transmission processes do not occur, enzyme activities were seen to go up 4 times and 2.4 times, respectively. Based on protein, enzyme activities in both brain material and dorsal root ganglia showed a similar pattern, whereas the activity increase in superior cervical ganglia was somewhat gradual (1.4 times from day 6 to day 100). Postnatal changes in gamma-GTP activities indicated a functional correlation with the maturation of amino acidergic structures. Kainic acid added to hippocampal extracts (0.45 and 5.0 mM) and, for comparison, to those of kidney (5.0 mM) did not yield any statistically significant effect on gamma-GTP activity.

Zusammenfassung Mit Hilfe einer speziellen Tetrazoliumsalz-Methodik wurde nachgewiesen, das zwischen der Na + -Konzentration im Inkubationsmedium und der Aktivitat der Glutamatdehydrogenase (GDH) im glutamatergen Neuropil, z. B. der Hippocampus-Formation oder des Cerebellums, eine auffallige Korrelation besteht; 130 bis 150 mmol Na + /1 bewirken eine maximale Formazan-produktion. An GDH-reaktiven Strukturen auserhalb des Neuropils sowie bei biochemischem Nachweis der GDH-Aktivitat in Hirnextrakten ist eine solche Natriumabhangigkeit nicht feststellbar. Die unter Na + -Einflus in glutamatergen Strukturarealen zu beobachtende Erhohung des Substratumsatzes last sich zwanglos mit den dort lokalisierten Na + -abhangigen Glutamat-aufnahmemechanismen erklaren.

The activities of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GIDH), the major glutamate metabolizing enzymes, were studied in hippocampal formation, cerebellar cortex, dorsal root ganglia, superior cervical ganglia and liver as a function of postnatal development. At birth, in all these nervous tissues the enzyme activities were quite low and showed similar levels (AAT 7–15 U/g wet weight; 0.18–0.23 U/mg protein; GIDH 3.4–13 U/g wet weight; 0.07–0.18 U/mg protein). Based on protein, AAT activity increased during the postnatal period studied 5.8 and 3.8 times in the hippocampal formation and cerebellar cortex, respectively, while the respective GIDH rise was 5.2 and 2.3 times. During postnatal maturation, enzyme activities in dorsal root ganglia showed only minor changes. In superior cervical ganglia, AAT and GIDH were remarkably constant. In liver the enzyme activities changed during postnatal development, but the activity curve profile was quite distinct from those obtained for brain regions. The steep rise of AAT and GIDH activities in brain regions is discussed as being a consequence of the maturation of preferably glutamatergic structures. Glutamatergic transmission processes obviously do not take place in superior cervical ganglia and dorsal root ganglia, and certainly not in liver. The present results suggest a quantitatively significant participation of glutamate transmitter metabolism in proportion to the whole glutamate metabolism of the CNS.

1. Transmitter glutamate/aspartate synthesis is known to proceed along different metabolic pathways. In this light, the functional relevance of glutamate dehydrogenase in postnatally maturing glutamatergic/aspartatergic structures was studied by means of quantitative enzyme histochemistry. 2. The basic requirements concerning the kinetics and calibration of the histochemical glutamate dehydrogenase reaction used were proved to be met in order to obtain valid quantitative data. 3. The histochemically demonstrable activity of glutamate dehydrogenase (EC 1.4.1.3) in the hippocampal formation of the rat increased markedly during postnatal development. On day 30, the distribution pattern observed was similar to that in adult animals. 4. While the enzyme activity rose within cell body layers from day 0 to day 30 by 240–285%, the increase in neuropil layers was found to be up to 830%. Maximum values were seen in the stratum lacunosum-moleculare of CA1 and CA3 and the stratum moleculare of the dentate fascia on day 30. 5. Since the hippocampal neuropil is supposed to be copiously provided with glutamatergic (and aspartatergic?) structures which become functional in rats during the first weeks of postnatal life, the increase in enzyme activity is discussed to be primarily a consequence of maturing synaptic systems using glutamate and/or aspartate as transmitters.

To study possible functional involvement of γ-glutamyl transpeptidase (GGT) in glutamate transmitter metabolism we lesioned putative glutamatergic structures of the rat hippocampal formation by intracerebroventricular (i.c.v.) injection of kainic acid (KA) or by surgical CA3 axotomy. Unilateral injection of KA into the left lateral cerebral ventricle of 30-day-old rats resulted in decreased GGT activity in hippocampal areas CA3, CA1 ipsilaterally, and in the contralateral area CA1, four hours after the induction of the chemical lesion. Four days after the injection, the enzyme activity was decreased in all hippocampal areas with the exception of the contralateral dentate gyrus. Four days after bilateral i.c.v. injection of KA, lower GGT levels were found than was seen after bilateral surgical lesion of the CA3 pyramidal cell axons (Schaffer's collaterals). The surgical lesion was followed by a decrease of GGT only in the stratum pyramidale and stratum radiatum of area CA1. In contrast to the effects in 30-day-old rats, unilateral i.c.v. injection of KA on postnatal day 12 did not alter the GGT activity in any studied hippocampal area presumably because of incomplete maturation of structures required for KA vulnerability.