Cytospectrophotometric study of the kinetics of histochemical reaction of GABA-transaminase in cryostat sections of rat cerebellar cortex

Summary A modification of the histochemical method for demonstration of GABA-transaminase is proposed. Substrate and cofactor concentrations are chosen on the basis of a kinetic investigation in cryostat sections of the rat cerebellar cortex. Enzymatic reactions were measured by quantitative microspectrophotometry. Michaelis constants for -oxoglutarate in the Purkinje cell layer and granular layer (K_m 1.7×10^–3 M) and white matter (K_m 3,8×10^–3 M) are found. It is shown that -oxoglutarate in concentrations higher than 5.2×10^–3 M (1 mg/ml) suppresses the reaction in sections by competitive inhibition. The advisability of addition of malonate, PMS and cyanide to the incubation medium is confirmed. It is suggested that there are some isoenzymes of GABA-transaminase with predominant localization either in neurons or glia.     

Segmental distribution and gestational changes of GABA-transaminase activity in the rat oviduct

The occurrence and the localization of 4-aminobutyrate:2-oxoglutarate transaminase (GABA-transaminase) in the non-pregnant and pregnant rat oviduct were examined using biochemical and enzyme histochemical techniques. Specific GABA-transaminase activity was detected in the ampullary and isthmic portions of the oviduct as well as in the utero-tubal junction. The enzymic activity was lower in the ampullary than in the isthmic or intramural segments of the oviduct. Pregnancy induced a significant increase of GABA-transaminase activity in each portion of the oviduct. Enzyme histochemistry showed the highest GABA-transaminase reactivity at the level of the epithelial cells of the oviduct irrespective of the portion of the tube examined. A faint specific activity was demonstrated in the smooth muscle of the oviduct while the serosa did not show specific staining. Our findings indicate that: the observed increase of GABA-transaminase activity in the oviduct of the pregnant rat may be responsible for the reduced GABA levels in the oviduct during gestation; and the extraneuronal localization of GABA-transaminase activity does not seem to support the suggestion of a possible GABAergic innervation of the oviduct.     

The histochemical demonstration of aniline hydroxylase activity in rat liver

Synopsis A procedure is described for the histochemical demonstration of aniline hydroxylase activity in cryostat sections of rat liver. Tissue sections are incubated in a medium containing aniline; thep-aminophenol formed as a result of enzymatic action is coupledin situ with Fast Blue RR. The staining reaction is found to be confined to the cytoplasm of the hepatocytes. Confirmatory tests for true enzymatic staining reaction include the incubation of sections in medium from which aniline is omitted, and under conditions of enzyme inhibibition. A method for the quantitation of the histochemical staining reaction is also described.The histochemical reactions have been investigated on rat livers subjected to conditions eliciting microsomal enzyme stimulation and inhibition, bothin vitro andin vivo. A close correlation was found between the staining reactions observed and the results of the quantitative histochemical method and the biochemical estimations of aniline hydroxylase activity in liver microsomal fractions obtained by differential centrifugation.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Histochemical technique for the demonstration of pyruvate kinase activity

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.     

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity

Synopsis Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigatingin vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.     

Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction

Dynamic analysis of the histochemical reaction of Karnovsky-Roots for acetylcholinesterase activity (AChE) is reported. Two methods were used. The first method was videography and densitometric analysis of frames from the film. The second method was direct microphotometric analysis of the reaction dynamics by the plug-method (measurement of average light transmission through a limited area of preparation or image). Special microchambers were used on the stage of an inverted microscope. The results showed the dynamics of final product accumulation in two structures of rat caudate nucleus: AChE-positive neuropil and the AChE-negative myelin bundle during histochemical incubation. Videography and densitometry of the digital images allowed morphologic and microphotometric analysis of changes in tissue sections during incubation, and the dynamic analysis permitted the study of enzyme kinetics in situ. Problems associated with microphotometric analysis of digital images for quantitative histochemical studies of the enzyme activity are discussed.     

Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction

Dynamic analysis of the histochemical reaction of Karnovsky-Roots for acetylcholinesterase activity (AChE) is reported. Two methods were used. The first method was videography and densitometric analysis of frames from the film. The second method was direct microphotometric analysis of the reaction dynamics by the plug-method (measurement of average light transmission through a limited area of preparation or image). Special microchambers were used on the stage of an inverted microscope. The results showed the dynamics of final product accumulation in two structures of rat caudate nucleus: AChE-positive neuropil and the AChE-negative myelin bundle during histochemical incubation. Videography and densitometry of the digital images allowed morphologic and microphotometric analysis of changes in tissue sections during incubation, and the dynamic analysis permitted the study of enzyme kinetics in situ. Problems associated with microphotometric analysis of digital images for quantitative histochemical studies of the enzyme activity are discussed.     

An improved histochemical method for measuring nitric oxide synthase activity

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section. Copyright © 1999 John Wiley & Sons, Ltd.     

Quantitative aspects of the histochemical tetrazolium salt reaction on monoamine oxidase activity in rat liver

Summary The tetrazolium method for the histochemical detection of monoamine oxidase (MAO) activity in rat liver cryostat sections has been tested for its specificity and its possible use in quantification. The tetrazolium salt tetranitro blue tetrazolium is recommended for the localization of MAO activity, rather than nitro blue tetrazolium or BPST [2-(2-benzothiazolyl)-3(4-phthalhydrazidyl)-5-styryl-tetrazolium]. Hardly any formazan was produced in the absence of the substrate tryptamine and Marsilid, a specific inhibitor of MAO activity, prevented formazan production almost completely. A linear relationship between the integrated absorbance measured with a microdensitometer and either the incubation period or section thickness was obtained. We conclude that the method described in this paper can be used for the quantitative analysis of MAO activity in tissue sections of rat liver. MAO activity was found to be 20–25% higher in the periportal zone of rat liver than in the perivenous zone.     

Histochemical technique for the demonstration of pyruvate kinase activity

Summary We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP andd-glucose to yieldd-glucose-6-phosphate and ADP. Thed-glucose-6-phosphate is oxidized by exogenous and endogenousd-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP⁺, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Schurin azide and amytal are included to block electron transfei to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal arusele. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.Dedicafed to Professor Dr. T.H. Schicbler on the occasion of his 65th birthday     

A quantitative histochemistry technique for measuring regional distribution of acetylcholinesterase in the brain using digital scanning densitometry

Studies of brain acetylcholinesterase (AChE) are traditionally based on biochemical assays, immunoreactivity, and histochemistry. Conventional histochemistry yields rich morphological data from tissue sections but yields quantitative results only with great difficulty. Several histochemical methods developed in recent years, including microdensitometry, microphotometry, and video-based histochemistry, are effective in quantitative and detailed study of AChE in tissue sections. However, they are usually time-consuming. As we report here, we adapted digital scanning densitometry to quantitate AChE histochemical staining in brain sections. The AChE and butyrylcholinesterase (BuChE), as measured by the method, were heterogeneously distributed throughout the brain, results that are consistent with those obtained by biochemical methods. The staining intensity is dependent on section thickness, substrate concentration, and reaction time. The cholinesterase inhibitor methyl paraoxon significantly decreased AChE staining intensity. Furthermore, data acquired from densitometry are similar to those obtained by video-based microscopy or by spectrophotometry. The advantage of the densitometric measurements compared to other quantitative histochemical methods is that it is very rapid while collecting data that are equivalent in quality. Because the digital scanning densitometers provide high quality and sensitive imaging, wide dynamic ranges, and convenient image analysis software, they are very useful tools in quantitative histochemistry.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S1/S2 segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 microns. Maximal DAP II activities were demonstrated at pH 5.5. The Km of DAP II was about 2.3 mM. In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S1/S2 segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation

Summary A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation.

A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.     

Light microscope study of the enzymatic activities of aspartate aminotransferase, GABA transaminase and acetylcholinesterase in the mesencephalic trigeminal nucleus neural complex of Mauremys caspica

The enzymatic activities of aspartate aminotransferase, GABA-transaminase and acetylcholinesterase were studied by means of histochemical methods in the mesencephalic trigeminal nucleus (MTN) neural complex of the turtle Mauremys caspica. Light microscope observations have demonstrated that MTN neurons have a positive reaction for these enzymes.     

Electrophoretic and histochemical studies of carbonic anhydrase activity

Summary A simple method for separation of carbonic anhydrase activity into components by electrophoresis on cellulose acetate strips is described. With this method, using barbiturate buffer systems at various pH values, two main components of CAH in rat erythrocytes, and the splitting of each of these into two minor components were revealed. Two components were also observed in the CAH activity in kidney and lens homogenates, and one component in brain homogenate. A modification of Häusler's histochemical method for CAH was adapted for visualization of the electrophoretically separated bands. This rendered the evalution of the results easier than with the quantitative measurements alone. The quantitative measurement of CAH activity in electrophoretic strips corresponded with the degree of staining by the histochemical method. This among other facts supports the view of the specificity of the histochemical method used. Some examples of the histochemical staining pattern of the CAH activity in rat tissues are given.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Quantitative microphotometric succinate dehydrogenase histochemistry in human nephron

A simple method for microphotometric evaluation of cryostat sections from human renal tissue routinely stained for succinate dehydrogenase activity by means of tetranitro-blue tetrazolium chloride is described and tested for validity. Manual absorbance measurement within single nephron segments from the same section allows to directly visualize the distribution pattern of this enzyme along the nephron. Photometric data can be expressed in relative enzyme activities by using the cortical collecting ducts within the same section as reference. This allows to compare measurements of different kidney sections stained by various incubation procedures. The agreement found between relative succinate dehydrogenase activities and recently published morphometric data on mitochondrial inner membranes along the rat nephron suggests that quantitative succinate dehydrogenase microphotometry is a useful histochemical tool for the assessment of renal mitochondrial cristae membranes.     

INHIBITION OF CHOLINE ACETYLTRANSFERASE AND ITS HISTOCHEMICAL LOCALIZATION

Abstract— A method for the histochemical identification of choline acetyltransferase has been investigated further by studying the effects of certain inhibitors of the enzyme both on rat brain homogenates and on the localization of the enzyme in tissue sections.
It was confirmed that acetyl-CoA hydrolase activity both in homogenates and in tissue sections is inhibited by preincubation in 1 mM-DFP. The effects of the choline acetyltransferase inhibitors chloro- and bromoacetylcholine on the appearance of histochemical staining were related to their activity in homogenates and tissue slices. Bromoketone was found to inhibit choline acetyltransferase in homogenates and, less efficiently, in tissue sections but it also inhibited the hydrolysis of acetyl-CoA by some other unknown enzyme which is inactivated by 1 mM-DFP.
The results obtained with the choline acetyltransferase inhibitors provide support for the specificity of the histochemical method.