Ultrastructural cytochemistry of p-N,N-dimethylamino-beta-phenethylamine (DAPA) oxidation reactions.

The ultracytochemical localization of amine oxidase (AO) activity is demonstrated with a new substrate, p-N,N-dimethylamino-beta-phenethylamine (DAPA). DAPA was designed to yield a stronger reducing agent on oxidation by monoamine oxidase (MAO) than is obtained from the MAO substrate, tryptamine, upon oxidation. Thus MAO and possibly other oxidase(s) can be demonstrated with DAPA and the tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). The latter is a nonosmiophilic tetrazolium salt which is reduced to an osmiophilic formazan. In addition, DAPA itself demonstrates AO activity ultracytochemically with and without BSPT. We speculate that either oxidative polymerization of DAPA or Schiff's base formation with protein after aldehyde formation is responsible for the latter reaction, which is made permanent for ultracytochemical localization by osmication at a later step. DAPA oxidation reaction products are demonstrated in guinea pig kidney, specifically in the endoplasmic reticulum, nuclear envelope and mitochondrial outer compartments and cristae. Differences in reaction product characteristics and localization in relation to formaldehyde fixation and the localization of reaction product in mitochondrial cristae, as well as outer compartments, suggest that DAPA oxidation is mediated through one or more MAOs and possible other oxidases.

The ultracytochemical demonstration of a formaldehyde-resistant dehydrogenase of leuco nitroxyl analogues.

A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.

Design of spindle poisons activated specifically by prostatic acid phosphatase (PAP) and new methods for PAP cytochemistry.

By taking advantage of the structural requirements of the substrates for prostatic acid phosphatase (PAP), which consist of steric hindrance and the presence of basic nitrogen in the molecule, potential cytotoxic agents (spindle poisons) are being designed that will become enzyme activated specifically by PAP. Colchicine has been converted to colchiceinamides of substituted ethanolamines and o-phosphoethanolamines. The rate of hydrolysis of the latter by human prostatic tissue as compared to the rate of hydrolysis by human kidneys (P/K ratio) is given and indicates a significant degree of specificity for PAP. Some preliminary toxicity data in mice are also given. New thiocolchicine derivatives with phosphates on ring B are also being prepared for study and some preliminary toxicity data are given. The observation in biochemical experiments that phosphorylcholine is a very specific substrate for PAP has led us to develop specific cytochemical methods for PAP for both light and electron microscopy. Preliminary observations are given and good evidence is provided that PAP is not a lysosomal enzyme, unlike other acid phosphatases. Furthermore, PAP is to other acid phosphatases what the cholinesterases are to other esterases. Since the acid phosphatase that is able to hydrolyze phosphorylcholine is characteristic of prostatic epithelium, this is the acid phosphatase that is referred to be the designation of PAP. Other acid phosphatases (both lysosomal and nonlysosomal) in prostatic epithelial cells are not demonstrated by this substrate and hence are not included in this designation.

Ultrastructural demonstration of cytochrome oxidase activity by the Nadi reaction with osmiophilic reagents.

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).