Hans Luppa

Cytochemical investigations on the localization of 5'-nucleotidase in the rat hippocampus with special reference to synaptic regions.

SummaryThe fine structural localization of the 5′-nucleotidase was investigated in the CA3 region of the rat hippocampus. The attention was focussed on the occurrence of the enzyme in the synaptic region. The 5′-nucleotidase activity was demonstrated at the surface membranes of axons and dendrites. Prominent portions of enzyme activity were detectable in the nuclei and the nuclear envelope, whereas the cytoplasmic organelles were nearly devoid of reaction product. In synapses five types of 5′-nucleotidase localization were revealed. A participation of the enzyme in the process of neurotransmission is discussed.

Inhibition of acetylcholinesterase and butyrylcholinesterase by the organophosphorus insecticide methylparathion in the central nervous system of the golden hamster (Mesocricetus auratus).

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.

The multiple forms of brain acetylcholinesterase

SummaryMicro-polyacrylamide gradient electrophoresis followed by active staining is applied for the demonstration of the multiple forms of acetylcholinesterase. Among other advantages the very small samples that enable the analysis of well-defined brain material as well as the almost histochemical conditions of incubation enable its successful use in topochemical investigations of the multiple form pattern of brain acetylcholinesterase.The acetylcholinesterase of bovine nc. caudatus could be separated into 4 multiple forms and the pattern was analysed microdensitometrically. These forms differ in their molecular weight as well as well as in their degree of membrane binding. Increasing ionic strength (NaCl) is followed by changes in the pattern. This result is discussed as caused by aggregation of enzyme subunits.

Demonstration of acetylcholinesterase by semipermeable membrane technique: Estimation of soluble and fixation-labile portions in different regions of the central nervous system

SummaryA histochemical method for the demonstration of acetylcholinesterase using semipermeable membranes is described. This technique prevents any loss of enzyme activity caused by dissolution and/or fixation. The soluble and fixation-labile portions of acetylcholinesterase were estimated in several regions of the central nervous system of the rat and differences were found. The method improves the accuracy of the histochemical demonstration of the acetylcholinesterase on the light microscopical level.

Multiple Localizations of Adenylate Cyclase in Rat Hippocampus

SummaryA new method for the histochemical demonstration of adenylate cyclase activity, introduced and biochemically tested by Poeggel et al. (1981a), was employed in nervous tissue. Using this method a multiple pattern of activity was detectable. Activity occurs in nervous as well as glial elements. Biochemical results and physiological conclusions could be confirmed by ultrahistochemical visualization of adenylate cyclase activity in nervous tissue. The specificity of the reaction is controlled by a number of variations of the incubation methods.

Specific demonstration of rat brain adenylate cyclase in polyacryl amide microgels by a new histochemical procedure

SummaryAdenylate cyclase from rat hippocampus was separated by electrophoresis in polyacryl amide microgels and stained for enzymatic activity using a new histochemical procedure. This method involves the use of AMP-PNP, aminophylline, dithiotreitole, and Sr2+ as “primary” capture ions, thus fulfilling all the demands for a really specific histochemical incubation medium for the enzyme. The incubation of the gels with this medium resulted in the inhibition of other enzymes, which are capable of splitting AMP-PNP (ATP: pyrophosphatase, alkaline phosphatase), whereas adenylate cyclase remained highly active under these conditions. The enzyme was found to be present in two forms in the gels. Both protein bands were stimulated by the addition of various biogenic amines to the incubation medium. One protein band was fully GMP-PNP dependent in its activity. It is reasonable to suppose that these forms are either differently high aggregated molecules of the enzyme or enzyme molecules bound to their regulatory sites.

Failure to demonstrate rat hippocampus adenylate cyclase.

SummaryThe fate of ATP exposed to rat hippocampal extracts was investigated after their separation by a microdisc electrophoresis technique. It could be demonstrated that the histochemical adenylate cyclase procedure using ATP as substrate is not suitable for specific localization of the enzyme, since other ATP hydrolysing enzymes were also able to convert ATP unless the concentrations of inhibitors reached 1 mM (ouabain) and 40 mM (NaF). With a prolonged incubation time of 18 h further substrate splitting protein zones could be revealed, possibly reflecting activities of enzymes involved in the hydrolysis of degradation products of ATP.

Pattern of NADPH-diaphorase active neurons in rat forebrain is unchanged after pentylenetetrazol kindling

The activity of NADPH-diaphorase in rat telencephalic structures has been revealed by use of a histochemical method. Multiple neurons belonging to different nuclei were found to contain the enzyme. Furthermore, diaphorase reactive nerve fibres and terminal fields were observed to be widely distributed throughout rat brain. Chemical kindling induced by pentylenetetrazol (PTZ) did not effect the regional distribution and cellular localization of the enzyme in the rat CNS.

Lokalisation und verhalten von hydrolasen und transmitterenzymen im dorsalen Corpus geniculatum laterale der ratte nach unilateraler enucleation

In the Corpus geniculatum laterale [pars dorsalis] (Cgl d) of the rat the distribution of the following enzymes is described on the light microscopical level: acid phosphatase, nonspecific esterases, Thiaminepyrophosphatase (TPPase), cholinesterases (AchE and ChE), GABA-alpha-ketoglutarat-transaminase (GABA-T), monoaminooxidase (MAO) and leucin-aminopeptidase. The acid phosphatase and TPPase are localized in the pericarya of different cell types of the Cgl d. The nonspecific esterases are present intracellularly and in the neuropil. With the GABA-T-reaction single cells of Cgl d are stained. The leucine aminopeptidase could not be demonstrated in the Cgl. The nonspecific cholinesterase is localized in the vessel walls. AChE and MAO are present in different fiber structures in the Cgl. Electron microscopical investigations of AChE showed a localization of the enzym in the extracellular room, intraaxonal and in the synaptic cleft. Separation of ChE in polyacrylamid gel resulted in 2 bands for the AChE and 1 band for the nonspecific ChE. After left side enucleation the reaction of the enzymes acid phosphatase, nonspecific esterases cholinesterases and TPPase was proofed topochemically at the section and biochemically at the homogenate. A decrease of enzyme activity in the enzymes acid phosphatase, nonspecific esterases and AchE is shown. The alterations are discussed in connection with the transneuronal degeneration.

Histochemische und biochemische Untersuchungen am Hippocampus und Neocortex der Wistarratte: I. Carboxylsäureesterasen, Transmitterenzyme und Transmitter im Normaltier1)2)

Some enzymatic parameters of neuronal transmission as well as the occurrence and the properties or carboxylic ester hydrolases in the hippocampal region of the wistar rat are investigated by histochemical and comparable biochemical methods. The acetylcholinesterase-, the monoamine oxidase- and the GABA-transaminase reaction are found at fibre structures, the course of which is seen more or less clearly. The histochemical picture of these enzymes is very different in each hippocampal layer and mainly limited by the corresponding number of reacting fibres. The origin and attribution of the fibres to the afferent and efferent systems are discussed. The occurrence of the acetylcholinesterase, the monoamine oxidase and the GABA-transferase as well as of the biogenic amines and the GABA are hints for the existence of cholinergic as well as aminergic and GABA-ergic processes of transmission in the hippocampal region. In the hippocampal region, the cingular and the optic cortex carboxylic ester hydrolases acetylcholinesterase, unspecific cholinesterase and the A-, B- and C-esterase could be demonstrated. The acetylcholinesterase of the hippocampal region is for the most part firmly membrane-bound and exists at least in two multiple, formalin-sensitive forms which are histochemically located in fibre structures. The unspecific cholinesterase, localized in the hippocampal region within vessel and capillary walls, exists in an electrophoretic mobile, formalin-sensitive form. Nearly half of the enzymes is soluble. A preferred binding to definite cell organelles was not demonstrable. In the hippocampal region the 3 multiple forms of the A-esterase are formalin-instable lyoenzymes. Good solubility and high formalin-sensitivity are the reason, why A-esterases are not demonstrable with usually histochemical methods. In the hippo ampal region the B-esterase is tightly bound to n electrophoretic mobile formalin-sensitive form in the microsomal fraction. In the cytoplasm of the neurones the desmoenzyme appears more or less granular. The 3 multiple forms of the C-esterase are formalin-sensitive to a different degree. Good solubility and low formalin-sensitivity, compared to the A-esterases are responsible for the fact, that the C-esterases can be shown histochemically only after en-bloc-fixation. The reaction products are granular. The similar behaviour of C-esterase and acid phosphatase, stated by many tests, suggests the C-esterases of the B- and C-type results in the same reactivity of pyramidal and granular cells of the hippocampal region. Some small, very strongly reacting cells belong to other cell types (probably basket cells or polymorphic cells).