Acta histochem. (lena) 95, 203- 219 (1993)Gustav Fischer Verlag lena' Stuttgart· New York

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Light microscopic visualization of monoamine oxidase using a ceriummethod

Georgios Nakos and Reinhart Gossrau

Department of Anatomy, Free University of Berlin, Konigin-Luise-Strafle 15, D-14195 Berlin, Germany

Accepted 2 August 1993

Summary

H20 rgenerating monoamine oxidase can be visualized in the light microscope withtetrazolium, metal salt (ferricyanide) and coupled peroxidatic oxidation methods. Due tomethodological draw-backs these procedures do no allow satisfactory results. In searchfor an alternative method a light microscopic cerium procedure was designed in whichthe primary reaction product, cerium perhydroxide, serves for the generation of am­plified and intensified diaminobenzidine brown. With this cerium-diaminobenzidine­H202-CO method monoamine oxidase was visualized more easily and reliably and withhigher sensitivity and more precise localization than with the other techniques. At pre­sent this method is considered to be the procedure of choice and was used to re-in­vestigate and investigate the distribution of monoamine oxidase in rats, mice, gerbils,guinea-pigs, marmosets, monkeys and man. In these species many cells and tissuesshowed monoamine oxidase activity where the enzyme has not yet been found before andthe structures with already known monoamine oxidase activity showed an improved lo­calization.

Key words: monoamine oxidase - cerium method - rat - mouse - gerbil - guinea­pig - marmoset monkey

Introduction

During our studies on the qualitative and quantitative light microscopic histochemis­try of reactive oxygen species-generating oxidases in healthy and diseased tissues therewas a need to deal also with the H202-generating monoamine oxidase. The enzyme canalready be visualized in the light microscope with tetrazolium (Glenner et al. 1957),coupled peroxidatic oxidation (Graham and Karnovsky, 1965; Ryder et al., 1980) andferricyanide methods (Hanker et al., 1973). However, all these procedures have theirdrawbacks and are therefore less suited for the investigation of monoamine oxidase(Lojda et al., 1979; Gossrau et al., 1991a; Robinson et al., 1991; Wohlrab and Gossrau,1992). By contrast cerium ions have been shown to be useful not only for oxidase ultracy­tochemistry (Briggs et al., 1975; Angermuller, 1989) but also for the light microscopic

Correspondence to: R. Gossrau

204 G. Nakos and R. Gossrau

histochemistry of various of H20rgenerating oxidases (Angermuller and Fahimi, 1988a,b; Gossrau et al., 1989; Halbhuber et al., 1991).

In this communication we describe a new light microscopic cerium procedure formonoamine oxidase A and B and report on the distribution of these enzymes in nervousand extranervous tissues of several mammalian species including man. Some preliminarydata about the enzyme using our first version of the cerium method have already beenpublished (Gossrau et al., 1991 a, b).

Material and Methods

Animals, tissue pretreatment: Adult Wistar rats (body weight 150-2oog), NMRI mice (body weight20- 25g), gerbils (body weight 30- 40g), guinea-pigs (body weight 500- 600g) and marmosets (bodyweight 300- 400g) of both sexes and bred on the premises were kept in Macrolon cages (rats, mice, ger­bils, guinea-pigs) or metal cages (marmosets) at standardized conditions (arteficial light-dark change;light from 7 a.m. to 7 p.m.; 22 ± I "C) with free access to Altromin rat, mouse, gerbil, guinea-pig or mar­moset diet (Altromin, Lippe, FRO) and tap water. The animals were sacrificed under Nembutal anaes­thesia between lOa.m. and 11a.m., and the following organs and tissues were removed quickly: Cer­ebrum, cerebellum, spinal cord, submandibular, sublingual and extraorbital gland, tongue, thymus,aorta, superior and inferior vena cava, esophagus, lung, heart, liver, fore- and hindstomach, pancreas,spleen, duodenum, jejunum, ileum, colon, rectum, adrenal gland, kidney, ureter, lymph nodes, urinarybladder, ovary, Fallopian tube, uterus, vagina, testis, epididymis, ductus deferens, as well as abdominal,fore- and hindleg skeletal muscles.

Samples of these tissues were mounted together on cork-plates with wet filter paper, wrapped withplastic foil, frozen in liquid N2 and stored in sealed plastic bags at - 25°C until use. 5 or 10urn sectionswere cut on a cryostat (model 2800N, Reichert-Jung, NuBloch, FRO) at - 25°C. Sections were eitherair-dried (unfixed sections), pretreated with acetone for 5 min at 4 °C or - 25°C or fixed with 0.1 to 2 070glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0 in the presence or absence of polyvinyl alcohol (5 to20010 PYA; M, = 10.000; Sigma, Heidelberg, FRO) or 5 to 15 0J0 sucrose (Serva, Heidelberg, FRO) for 1to IOmin at 4°C. The glutaraldehyde-fixed sections were rinsed in tap water for 5 min and in distilledwater for another 5 min.

Incubation media, incubation techniques. Aqueous incubation media or incubation media containing5 0J0 to 20 0J0 PYA (M, = 10.000) were used. The incubation media had the following composition: 0.1 Mcacodylate, Tris-HCI, Tris-maleate or PIPES buffer, pH 7.4, 0.1 to 30mM CeCI3, 0.1 M NaN3 or amino­triazole and 1 to 50mM tryptamine, tyramine, histamine, 5-hydroxytryptamine (serotonine),phenylethylamine, dopamine, adrenaline or noradrenaline as substrate. With aequous media incubationwas performed in cuvettes in a shaking water bath for 5 to 60min at 37°C. PYA media were poured intowells made by Perspex rings surrounding the sections and fixed on glass-slides with vaseline and in­cubated for the same time and at the same temperature.

After incubation the media were removed; the sections were rinsed in tap water and then in distilledwater.

Visualization ofcerium perhydroxide generated by the enzyme was performed according to Angermiillerand Fahimi (1988a, b) as modified by Oossrau et al, (1989)using Co2+ ions for the amplification and/orintensification of diaminobenzidine (DAB) brown. After rinsing in tap water and rinsing in distilledwater, the sections were mounted in glycerol jelly and evaluated and photographed using a Zeiss photo­microscope with an automatic camera.

Control reactions were performed in the absence of the substrate, in the presence of the substrate and theMAOX inhibitors pargyline, clorgyline or iproniazide each in concentrations between 0.1 and 5 mM andin the absence of oxygen (by bubbling N2through the media), in the presence of 0.5 0J0 or I 0J0 (w/v) cat­alase (bovine liver, approx. 20.oooU/mg enzyme, Serva) instead of NaN3 or aminotriazole or in theabsence of Ce3+ ions as H20 2trapping agent.

As further controls benzylamine oxidase (BAOX, semicarbazidesensitive oxidase, EC no. not yet as­signed) was visualized with the cerium method according to Nakos and Oossrau (1993a) with benzyl-

Cerium method for monoamine oxidase 205

amine (Sigma) as substrate in Tris-HCI buffer, pH 7.4 in the absence or presence of 1 to 5mM semicar­bazide as was diamine oxidase (DAOX, histaminase, polyamine oxidase, EC 1.4.3.6) with a corre­sponding cerium procedure according to Nakos and Gossrau (1993b) using cadaverine, putrescine, sper­midine or spermine (Sigma) as substrates and 1mM semicarbazide or 1mM aminoguanidine (Sigma) asinhibitors.

Chemicals, if not further specified, were obtained from Merck (Darmstadt, FRO), Serva (Heidelberg,FRO), and Sigma (Munich, FRO).

Results

Methodological experiments

Tissue pretreatment, incubation media. Unfixed (air-dried) or acetone-pretreatedcryosections enabled high amounts of stain which, however, was not always precisely lo­calized. Precisely localized final reaction product in identical amounts was seen in freshsections when the incubation media contained 5 % or 10% PYA; lower quantities ofstain were present when using 15% or 20 % PYA. Independent of the PYA concentra­tion the background staining was absent or considerably lower than with aequous in­cubation media. Furthermore, PVA-containing media reduced or prevented nonspecificnuclear staining and improved the preservation of tissue structure. Localization of thereaction product and preservation of tissue structure was also improved and clean sec­tions were obtained when 15% or 20 % PYA were present in the glutaraldehyde fixative;less satisfactory were the results with a PVA concentration of 1 %, 5 % or 10%. 0.1 to1% Glutaraldehyde in cacodylate, Tris-maleate, Tris-HCl or phosphate buffer preservedtissue structure equally well and delivered similar amounts of stain; less stain was foundafter fixation in 1.5 or 2 % glutaraldehyde. Presence of 5, 10 or 15 % sucrose in the fixa­tive may yield higher amounts of stain. Variation of the fixation time between 1 to 10mindid not affect the quantity of final rection product significantly.

Incubation times, section thickness. Over a period of 30min 5 urn sections delivered ap­proximately half the amount of stain as 10 urn sections with substrate concentrations of5 mM tryptamine or tyramine.

Cerium concentration. Up to a cerium concentration of 1mM increasing quantities ofamplified and intensified DAB brown were found. Concentrations between 2 and 10mMdid not enhance staining intensity, and higher concentrations inhibited MAOX activity.

Substrates, substrate concentrations. Species-independently, tyramine or tryptamineyielded the highest amounts of stain followed by phenylethylamine and serotonine. Do­pamine, adrenaline, histamine and noradrenaline were not oxidized in visible amounts.Up to 10mM substrate no inhibition was found; substrate concentrations of 1 to 2mMenabled maximum staining intensity.

Buffers. In the presence of Tris-HCl far higher amounts of stain were produced thanwith Tris-maleate or cacodylate. When Tris-HCl was replaced by PIPES similar stainingintensity was obtained and the background staining was somewhat lower.

Control reactions. Negative results were obtained in the absence of substrates, Ce3+ ionsor oxygen or in the presence of catalase in incubation media without azide. An exceptionwere granulocytes which showed a positive reaction in media free of substrate or Ce3+(caused by the reaction of myeloperoxidase and/or eosinophil peroxidase which use the

206 G. Nakos and R. Gossrau

visualization solution as incubation medium and are not inhibited by azide). As MAOX,BAOX was present in smooth muscle cells in the walls of blood vesselsand hollow organsbut mostly with higher staining intensity. Dependent of the site and species this stainingwas suppressed or abolished in the presence of the BAOX inhibitor semicarbazide butnot always in that of MAOX inhibitors (Nakos and Gossrau 1993a). DAOX could befound in structures different from MAOX and staining was prevented in the presence ofthe DAOX inhibitors semicarbazide or aminoguanidine but not by MAOX inhibitors(Nakos and Gossrau, 1993 b).

Inhibition tests. Using 5!tm sections the MAOX A and B inhibitors iproniazide or par­gyline suppressed or abolished enzyme activity depending on the inhibitor concentration,the speciesand the organ or tissue. Reliable and general responses were found with 0.1 or0.5 mM concentrations; 0.01 mM concentration was less effective when lO!tm thickcryosections and simultaneous inhibition were used. In the presence of the MAOX Ainhibitor clorgyline in a concentration of 0.5 mM heart muscle tissue showed no longerenzyme activity (Table 1).

In conclusion, the following procedure appears to be optimal for the light mi­croscopic visualization of MAOX A and B: Fixation of cryosections with 0.5 010 glutar­aldehyde in 0.1 M cacodylate, phosphate, or tris-HCl buffer in the presence or absenceof 15% or 20 % PVA or use of unfixed air-dried sections if 5 % or 10% PVA are present

Table 1. Inhibition of monoamine oxidase in rat and mouse tissues by iproniazide, pargyline andclorgyline

Tissue activity in the presence of

iproniazide pargyline clorgyline

Submandibular 0 0 +glandThymus 0 0 +Lung 0 0 +Liver 0 0 +Stomach

fore 0 0 +hind 0 0 +

Intestinesmall 0 0 +large 0 0 +

Kidney 0 0 +Uterus 0 0 +Spleen + 0 +Heart + + 0Smooth muscle cells (+) (+) +Pancreas

endocrine 0 0 +Brain 0/+ 0/+ +

Fig. 1. Mouse, MAOX in cerebral neurons (arrows) and glial cells and their processes (ar­rowheads). Fig. 2. Rat, MAOX in glial cells (arrows). Arrowheads =glial cell processes, thin arrows =septum of glial cells. Fig. 3. Mouse, MAOX in pericarya (arrows) of the pancreas, a = acini; thin ar­row =arterous smooth muscle cells; d =excretory duct. Fig. 4. Mouse, MAOX in respiratory epithelialcells (arrows), arrowheads = surfactant cells. Objective X 40.

Cerium method for monoamine oxidase 207

208 G. Nakos and R. Gossrau

in the incubation media. Fixation is followed by rinsing for 5min in tap water and then indistilled water. Incubation in a solution of 0.1 M Tris-HCI, pH 7.6 containing 5mMCeCI3, 0.1 mM NaN3, 5mM tyramine , tryptamine, serotonine or phenylethylamine for 1to 60min at 37 °C followed by rinsing of the sections in tap water and then in distilledwater; visualization by a DAB, H202, Co and azide containing solution for 25min at37 °C as described by Gossrau et al. (1989).

Distribution of monoamine oxidase

Species-independent distribution . In the central nervous system MAOX was found innerve cells of the cerebrum (Fig. I), cerebellum and spinal cord as well as microvillous,ciliated and non-ciliated (tanycytes) ependymal cells, in glial cells (Fig. 2) of the grey andwhite matter and in capillary endothelial cells of the blood brain barrier. In the peri­pheral nervous system the enzyme reacted in nerve fibers and vegetative nerve cells(Fig. 3). In the respiratory tract (Fig. 4) ciliated and/or microvillous cells were positivefor the enzyme, and in the digestive tract staining was present in small intestinal entero­cytes (Fig. 5, 6) as well as in hepatocytes (Fig.7). In addition , periportal hepatocytesproduced averagely more stain than those in the pericentral region. In the kidney, theenzyme was present in the tubular apparatus of all species; the type and number oftubules with reaction product differed, however, species-dependently. Leydig cells(Fig. 8) of the testis reacted for MAOX in rats, mice, marmosesets, quinea-pig and ger­bils with species-dependent activity differences ; the highest amount of stain was found ingerbils. In the digestive tract (Fig. 9, 10, 11), epididymis, myometrium (Fig. 13) andblood vessels (Fig.B, 13) smooth muscle cells expressed enzyme activity. The smoothmuscle cells of the ductus deferens were free of enzyme activity. - Strong activities ofMAOX were present in the visceral epithelial cells of the extraplacental yolk sac of ratsand mice (the yolk sac of the other species was not analysed).

Species-dependent distribution. The majority of tissues showed species-dependentMAOX activities and localizations. Epithelial cells of the choroid plexus (Fig. 14) werestained in mice but not in the other murine rodents. Also differently behaved glial cells inthe circumventriular organs, pineal gland, subcomissural, subfornical and paraventricu­lar organ, and median eminence and in the neurohypophyis . In the heart (Fig. 15)singleor groups of atrial and ventricular cardiomyocytes produced stain in rats; occasionally,differences in staining intensity occurred inside the same cardiomyocyte. In the lungsurfactant cells (type II pneumocytes; Fig. 16) were positive in mice and rats as wereparietal cells (Fig. 17) in the stomach of mice, gerbils and marmosets but not in rats. Inrats large intestinal enterocytes were free of the enzyme. In rat and mouse pancreatic isletcells (Fig. 18) MAOX was highly active; the endocrine cells in the islets of gerbils wereweakly stained; those of marmosets reacted only with tryptamine as substrate and thoseof guinea-pigs substrate-independently not at all. In the uterus subepithelial connectivetissue cells were stained in mice, surface epithelial cells in gerbils and capillary en­dothelial cells in rats where the capillary endothelium (sinusoidal cells) was also positivein corpora lutea of the ovary. In the same organ of gerbils granulosa cells (Fig. 19) wereheavily stained.

Fig.5 . Mouse MAOX in jejunal enterocytes (arrows); arrowheads = goblet cells. Fig. 6. Mouse,MAOX in colonic surface and crypt enterocytes (arrows). Arrowheads =goblet cells; m =muscula­ris. Fig. 7. Mouse, MAOX in hepatocytes (arrows). Arrowheads = sinusoids. Fig. 8. Gerbil, MAOX inLeydig cells (arrows). Thin arrows =arterous smooth muscle cells. Arrowheads =spermatides. Objectivex 40.

Cerium method for monoamine oxidase 209

210 G. Nakos and R. Gossrau

cc

Cerium method for monoamine oxidase 211

Although (see above) MAOX was present in the tubular apparatus of the kidney aspecies-dependent distribution of the enzyme in the different portions of the renaltubules was found. In rats and mice (Fig. 20,21) MAOX was seen in single or groups ofdistal tubular cells (shown by double incubation with non-specific alkaline phosphatase;Nakos and Gossrau, unpublished observations) while in marmosets and guinea-pigsepithelial cells of the convoluted and straight proximal and distal tubules contained reac­tion product. The gerbil kidney showed final reaction product in the epithelium of thestraight proximal tubules. In addition, MAOX was detected in certain epithelial cells inthe collecting tubules (Fig. 22) of the medulla (revealed by double incubation with non­specific esterase; Nakos and Gossrau, unpublished data) in mice.

In the placenta MAOX was present with lower activity in the trophoblast of themouse labyrinth and with high activity in the syncytiotrophoblast of the human placenta.

Discussion

So far the catalytic properties of H202-generating monoamine oxidase could be usedto visualize the enzyme in the light microscope with tetrazolium salt (Glenner et al.,1957), metal salt (ferricyanide procedure; Hanker et al., 1973) and coupled peroxidaticmethods (Graham and Karnovsky, 1966; Ryder et al., 1980). With the cerium-DAB­H202-CO technique a further procedure is now available to study the enzyme light mi­croscopically.

The cerium method is free of some of the drawbacks the other procedures have. Inde­pendent of the type of salt used the disadvantages of the tetrazolium salt reaction are theunexplained reaction mechanism, the autooxidation of some of the substrates by thetetrazolium salts (Lojda et al., 1979; Robinson et al., 1991; Wohlrab and Gossrau, 1992)and the different potential of the aldehydes generated by the enzyme and needed for thereduction of the tetrazolium salts (Kashimoto et al., 1983). Furthermore, certainMAOX-containing structures, e. g. glial cells, B cells of the exocrine pancreas or type 11­pneumocytes cannot be detected with the tetrazolium salt procedure. Similar reductionproblems may arise with the metal salt procedure using ferricyanide as reducing agent;furthermore, ferricyanide is presumed to inhibit monoamine oxidase at a comparativelylarge extent (Robinson et al., 1991; Wohlrab and Gossrau, 1992).

Due to the unexplained reaction mechanism of the tetrazolium salt and ferricyanideprocedure it is also unknown how far the three reaction products, aldehyde, H202 andammonium ions contribute to the final reaction product. This is known, however, for thecoupled peroxidatic method and cerium-DAB-H20rCo technique since both proceduresuse the chemically defined H202 produced by monoamine oxidase for the generation ofthe final reaction product. However, in the coupled peroxidatic method where DAB (oranother peroxidase substrate) is used as reducing agent for H202, DAB serves as subs­trate also for cytochrome C oxidase and granulocyte peroxidases and can be oxidized bynon-enzymatic hemoproteins, and these compounds cannot always be blocked reliablyby inhibitors (Lojda et al., 1979; Deimann et al., 1991; Gossrau et al., 1993).

Fig. 9. Mouse, MAOX in smooth muscle cells of the inner (double arrow) and outer layer (arrows) of thegastric muscularis. Fig. 10. Mouse, MAOX in smooth muscle cells of the inner (double arrows) andouter layer (arrows) of the muscularis of the ileum. Arrowhead = MAOX in differentiating enteroblastslenterocytes. c = crypts. Fig. 11. Rat, MAOX in smooth muscle cells of the inner (double arrow) andouter layer (arrow) of the colonic muscularis. Thin arrows = smooth muscle cells of the muscularis mu­cosae. Arrowheads = coraction of peroxidase in granuloxytes, Fig. 12. Rat, MAOX in the smoothmuscle cells (arrows) of the muscular layer of the epididymal duct. Arrowheads =principal cells. s =sperms in the duct lumen. Objective x 40.

212 G. Nakos and R. Gossrau

Cerium method for monoamine oxidase 213

In the coupled peroxidatic method exogenous peroxidase is used as auxiliary (ex­ogenous) enzyme. This may include limited penetration or diffusion of peroxidase to thesite of HzOz production and may result in less precise localization. Additionally, forproper results this method requires fixation by perfusion which means inhibition of thefixation-sensitive monoamine oxidase. This is also shown by the rather weak staining ac­tivity of glial cells which can contain considerable activities of the enzyme and by itsabsence in brain capillary endothelial cells using this procedure (Kashimoto et al., 1983;Konradi et al., 1989). Another disadvantage is that floating incubation is recommendedfor optimal results, a troublesome, time-consuming and less effective technique espe­cially when many different tissues have to be handled simultaneously.

By contrast, in the cerium method a very mild or no fixation is used and the Ce3+ions (instead of the peroxidase-DAB system) trap HzOz effectively (as shown by ultra­cytochemistry for many HzOz-generating oxidases including monoamine oxidase; Briggset al., 1975; Fujimoto et al., 1982; Angermuller, 1989; Robinson et al., 1991; Wohlraband Gossrau, 1992). Furthermore, after proper mounting of small pieces practically allorgans of one species can be incubated and microscoped together. Consequently, morepositive sites with more precise localization of the final reaction product can easily be re­vealed simultaneously in nervous and extranervous tissue with the cerium-DAB-HzOz­Co method. Therefore, at present this procedure can be considered as the light mi­croscopic method of choice for the qualitative investigation of monoamine oxidase. Fi­nally, the cerium technique is cheaper than the coupled peroxidatic oxidation method.The tetrazolium and metal salt procedures are inferior to the other two methods andshould no longer be used.

Application of the cerium-DAB-HzOz procedure to different species reveals species­independent and species-dependent activities and distribution patterns; the species­dependence dominates. This shows that functions related to monoamine oxidase, i. e.degradation (clearance, scavenging, inactivation, removal) of exogenous and en­dogenous non-biogenic and biogenic amines including transmitters such as serotonine(Robinson et al., 1991)can be performed by different cells in the concerned tissues andorgans. Alternatively, the enzyme may be present species-independently but cannot beproperly visualized for various reasons including methodological ones. However,biochemical measurements of monoamine oxidase (Gossrau et al., 1991 b) carried out inparallel to the histochemical studies with the cerium method and using fluorescent hy­droxyphenyl compounds (Guibault et al., 1966)have also shown considerable species-de­pendent monoamine oxidase activities (Gossrau, unpublished data) which suggests trueactivity differences.

Due to the complex and special situation of monoamine oxidase in the nervous tissueof rats, mice, gerbils, guinea-pigs and marmosets the enzyme will be described in detailand discussed separately.

With the exception of serotonine (5-hydroxytryptamine) the endogenous amines, a­drenaline, noradrenaline and dopamine are not oxidized (and also not when used in theother methods; Gossrau and Nakos, unpublished observations) in visible amounts andtyramine, tryptamine and phenylethylamine used for the histochemical demonstration,are primarily not endogenous substrates. This rises the question whether outside the

Fig. 13. Rat, staining of smooth muscle cells in the myometrium (arrows). Arrowheads = arteroussmooth muscle cells. Fig. 14. Mouse, MAOX in plexus choroid epithelial cells (arrows). Arrowheads =ependymal cells. Fig. 15. Rat, different staining of myocardiocytes (arrows) by MAOX. Fig. 16.Mouse, MAOX in type II-pneumocytes (arrows). Objective x 40.

214 G. Nakos and R. Gossrau

18 a

Cerium method for monoamine oxidase 215

epithelial cells at the border between the inner and outer milieu, i. e. in the trache­bronchial tree and digestive tract (see below) really the physiologically relevantmonoamine oxidase is visualized everywhere.

Monoamine oxidases are important H202 generators, a fact mostly neglected in theliterature where still the main emphasis is placed on the degradative deamination of theenzyme, and thus its regulatory function in biogenic amine including neurotransmittermetabolism (Oreland and Callingham, 1987; Riederer and Youduim, 1990). However,H202 represents either a potential oxyradical or a physiological intermediate product andboth should be considered when the function of the enzyme in nervous and extranervoustissue is discussed (Halliwell and Gutteridge, 1989; Callingham et aI., 1990).

Our study shows that the epithelial cells of the conducting portion of the respiratorytract and intestinal enterocytes have high activities of monoamine oxidases. They shouldtherefore be able to detoxify effectively aerial or nutritional harmful vasoactive aminessuch as tryptamine, phenylethylamine, tyramine or other amines in the food or air pre­venting blood pressure crisis (Schwenk, 1989; Forth and Henschler, 1992).

Because of this effective amine-degrading monoamine oxidase barrier in epithelialcells at the border between the outer and inner milieu critical concentrations of aminesshould not enter the body. Therefore, the other monoamine oxidase-containing struc­tures, such as cardiomyocytes, hepatocytes, renal tubular cells, smooth muscle cells orvascular endothelial cells may primarily oxidatively deaminate (degrade, inactivate, sca­venge, remove) endogenous amines. Unclear is the source of these compounds in extra­neural tissue. In aminergic neurons they are synthezised by the corresponding enzymessuch as phenylalanine and tyrosine hydroxylases, DOPA decarboxylase and hydroxylaseand phenylethanolamine methyltransferase (Iversen, 1975; Devlin, 1992). Whetherbiogenic amines can be synthesized and not only degraded (by monoamine oxidase andcatechol-O-methyltransferase) in nonneural cells in now investigated. Principally itshould be possible as shown for DOPA or histidine decarboxylase in extranervous tissues(Musacchio, 1975; Siraganian, 1988).

In the heart comparative fluorescence studies on aminergic innervation and his­tochemical monoamine oxidase activity and distribution have shown, that in guinea-pigsinnervation density and enzyme activity must not correlate (Gossrau and Nakos, unpub­lished data). The working myocardium of this species is especially rich in aminergic fi­bers (Winkler, 1969) but has a very low monoamine oxidase activity in the car­diomyocytes. Vice versa the rat working myocardium has a far less dense aminergic in­nervation (Winkler, 1969) but high activities of monoamine oxidase in itscardiomyocytes. Furthermore, the rat cardiomyocytes can be divided in different sub­populations. This contrasts with the assumption that working cardiomyocytes representan enzymatically homogenous cell population. From the guinea-pig and rat data one maysuggest a participation of the heart muscle cells in the degradation of extracardiallyproduced amines rather than that of the locally released transmitter substance noradren­aline.

Also those amines which are degraded by hepatocytes and tubular cells (of which typeso ever) appear to be of mostly extrahepatic and extrarenal origin because both organshave a minor sympathetic vegative innervation; by contrast the noradrenaline set free inthe muscularis (media) of blood vessel walls is presumably degraded (oxidized) by

Fig. 17. Mouse, MAOX in parietal cells (arrows) of the stomach. Arrowheads = chief cells. m = muscu­laris, mm=muscularis mucosae. Fig. IS. Mouse, MAOX in endocrine cells (arrows) of the pancreas.a = acini. Fig. 19. Gerbil, MAOX in granulosa cells (arrows). Fig. 20. Rat, MAOX in distal tubularcells of the cortex (arrows). g = glomerulus, p = proximal tubules. Objective X 40.

216 G. Nakos and R. Gossrau

monoamine oxidase in their smooth muscle cells (Iversen, 1975). However, also here thesituation seems to be more complicated since the smooth muscle cells of the ductus def­erens with a dense adrenergic innervation can be free of monoamine oxidase while thoseof the epididymal duct behave vice versa (Brandes, 1974).

Striking relative (the enzyme is principally present in all investigated animals) speciesdifferences occur in the kidney concerning type and number of monoamine oxidase-posi­tive tubular cells and therefore the total monoamine degrading capacity (the aminesthemselves should derive from the primary urine and/or the peritubular blood). In ratsand mice amine removal occurs in special (not all) parts of the convoluted distal tubule.This suggests a further functional subdivision and division of labor of the tubular appa­ratus and the degradation of luminal amines far away from their place of origin in theglomerulus or after their passage of nearly the whole nephron. Mice have additionalepithelial cells in the collecting ducts being able to detoxify amines; whether they corre­spond to the dark or light (intercalated) cells has to be cleared by further studies. By con­trast, the epithelial cells of the straight proximal tubules are responsible for amine uptakeand scavenging in gerbils and all portions of the tubular part of the nephrons are in­volved in these processes in guinea-pigs and marmosets.

, d

22Fig.21. Mouse, MAOX in epithelial cells of distal convoluted tubules (arrows). g = glomerulus, p = prox­imal tubules.Fig. 22. Mouse, MAOX in certain collecting duct cells (arrows). d = distal tubules. Objective x 40.

Cerium method for monoamine oxidase 217

Absolute species differences exist in the endocrine pancreas where monoamine ox­idase is present in mice and rats in high and in gerbils as well marmosets in medium orlow activities but not in guinea-pigs. Similar observations are true for parietal cells in thestomach, type I1-pneumocytes (surfactant cells), the epithelium, subepithelial connectivetissue and capillary endothelium of the uterus or in the ovary. Unclear is at present whichtypes of endocrine cells do contain the enzyme in the endocrine pancreas (and possiblyother organs), which is the physiological role of monoamine oxidase and whether there isa correlation between the enzyme-positive cells and those containing amine oxidase subs­trates, e. g. serotonine (Lange, 1973). In the islets most of them should represent B cellsbut the presence of monoamine oxidase in other cell types cannot be excluded. Thesurfactant cells are positive for the enzyme in mice and rats. The type Il-pneumocytes to­gether with the respiratory epithelial cells represent the single MAOX-positive structuresin the murine rodent lung. Whether they are therefore responsible for the clearance ofplasma amines (Tripton et al., 1976; Ganong, 1991) needs to be clarified since both celltypes lack direct contact with the blood. For the regulation of monoamine oxidase in thelung evidence is available that this may occur by aerial oxygen. In rats and mice beforebirth enzyme activity can only be found in respiratory epithelial cells. After birthmonoamine oxidase becomes active in the type I1-pneumocytes during the first two daysof life (Nakos and Gossrau, unpublished data). Activity in the capillary endotheliumoutside nervous tissue shows that monoamine oxidase does not represent an enzymespecific for capillary endothelial cells of the blood brain barrier (Vorbrodt, 1988) in thecentral nervous system. Further investigations have to find out whether these species dif­ferences are connected with physiologically relevant functional differences.

In conclusion, the cerium-DAB-H20rCo-procedure represents a histochemicalmethod by means of which monoamine oxidase can be better studied in the light mi­croscope than with the other procedures. Therefore, using this new technique additionalinformation will hopefully be possible about the cell, tissue and organ function of thisamine-degrading oxidase.

Acknowledgements

We are thankful to Ms. H. Richter for technical assistance, Ms. U. Sauerbier forphotographic work and Ms. A. Hechel for the preparation of the manuscript.

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