Ultracytochemical localization of guanylate cyclase activity in guinea-pig peritoneal macrophages under different physiological conditions

Summary Guanylate cyclase activity was investigated in guinea-pig peritoneal macrophages under different physiological conditions (such as adhesion and phagocytosis) with an ultracytochemical method using guanylyl-imidodiphosphate as a substrate. The enzyme was detected on the perinuclear envelope, endoplasmic reticulum, Golgi complex and mitochondria of adherent and phagocytozing macrophages. No reaction product was present around phagocytozed polystyrene particles. The amount of final reaction product was increased by the addition of sodium azide to the incubation medium and no staining was observed when the substrate was omitted from the medium.     

The cytochemical localization of cholinesterase activity in the developing chick heart

Summary The electronhistochemical localization of the cholinesterases of developing chick heart muscle cells has been studied with the aid of a substrate which incorporates an enzyme-susceptible thiolester group and a diazonium group into the same molecule. The embryonic chick heart exhibits cholinesterase activity from Hamilton-Hambruger stage 3 through to four days post hatching. Although enzyme activity is not demonstrated in every location at all stages studied, it has been observed on the nuclear envelope, golgi complex, rough and smooth endoplasmic reticulum, mitochondria and myofilaments. A change in the type of activity has been demonstrated, acetyl-cholinesterase is found during the first fourteen days of development but thereafter, non-specific cholinesterase is seen instead. As nerves have not been found in relation to the working myocardium, further support is given to the concept that an acetylcholine-cholinesterase system of myogenic origin is involved in spontaneous contraction. Consideration of the distribution of enzyme within the myocardial cell, raises the possibility that cholinesterase may be concerned in a regulatory mechanism of protein synthesis, a suggestion made previously in connection with liver cells.     

Ultracytochemical heterogeneity of cytochrome oxidase activity in rat cardiomyocytes

Optimum conditions are developed for ultracytochemical detection of cytochrome oxidase in cardiomyocytes from the rat heart ventricles. Conditions mentioned above rest on using of o-dianisidine and double contrasting of microscopic sections by uranyl and lead acetates. Data from studies in heterogeneity of cytochrome oxidase activity as dependent on the structural-functional state (electronic density) of myocardium cells are presented.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Histochemical demonstration of acetylcholinesterase in the hypothalamus of the female guinea-pig

Summary The distribution of acetylcholinesterase (AChE) in the hypothalamus of the female guinea pig has been examined histochemically.Activity was found in neurones of the supraoptic, paraventricular and infundibular nuclei; in the lateral hypothalamic area, in cells dorsomedial to the fornix at the level of the paraventricular nucleus and in a large group of cells surrounding the fornix at the level of the in-fundibular nucleus. A small well-stained group of cells, not identified in histological preparations, was present ventral to the paraventricular nucleus. The neuropil stained at the lateral border of the paraventricular nucleus and in the medial mammillary, suprachiasmatic and dorsomedial nuclei. The walls of some blood vessels stained; activity was particularly strong in vessels in the preoptic area and at the base of the median eminence. Nerve fibres on blood vessels ventral to the hypothalamus also stained.The degree of enzyme activity in the cell groups was compared in immature, pregnant, lactating, ovariectomized and hysterectomized animals. The greatest variation occurred in the infundibular nucleus. Hypophysectomy markedly reduced staining in the supraoptic nucleus. The possibility that AChE may be involved in the elaboration or transport of releasing factors is discussed.The authors are indebted to Dr. J. S. Perry for doing the surgery involved in this work. They are also grateful to him and to Dr. R. B. Heap for helpful discussions and to Miss M. Hamon for excellent technical assistance.     

Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles

A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate D-fructose-6-phosphate to D-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and D-glyceral-dehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-D-glycerate. Concomitantly the electrons are transported via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Histochemical demonstration of cholinesterase activity in the cornea of the rat and the effect of various denervations on the corneal nerves

Summary Cholinesterase (ChE) activities of the rat cornea were demonstrated histochemically by using both light and electron microscopes. Acetylcholinesterase (AChE) reaction was localized in the axolemma of the nerves in the corneal stroma. The epithelial cell membranes and the intraepithelial nerve endings also showed AChE reaction.Non-specific cholinesterase (NsChE) activity was observed only in the endothelial cell membranes.Cervical sympathectomy, ciliary ganglionectomy and stereotactic coagulation of the ophthalmic division of the trigeminal nerve were performed in order to study the routes of the AChE-containing nerves to the cornea. The disappearence of AChE-containing nerves was observed only after ophthalmic neurotomy. It is suggested that the AChE-containing nerves are distributed to the rat cornea exclusively via the ophthalmic nerve. They seem to be sensory nerves.     

Specific demonstration of acetylcholinesterase and non-specific cholinesterase in the adrenal gland of the rat

Summary Distribution of cholinesterase in the adrenal medulla of the rat was studied using acetylthiocholine, butyrylthiocholine and -naphtyl acetate as substrates and eserine, di-isopropylfluorophosphate (DFP), 1:5-bid-(4-trimethylammoniumphenyl)-pentan-3-one di-iodide (62. C. 47) and tetra-isopropylpyrophosphoramide (iso-OMPA) as inhibitors.Acetylcholinesterase was observed in the nerve trunks, the ganglion cells, the coarse and the fine nerve fibers. The fine medullary network showed along the fibers small strongly positive ovoid bodies.Non-specific cholinesterase was detected in the capsule, the nerve trunks, the coarse nerve fibers and the fibers surrounding the noradrenaline-containing, fluorescent medullary cell islets. A weak reaction was also seen in the cytoplasm of the medullary cells. The fine medullary fibers with the ovoid bodies were essentially negative.A method was developed to demonstrate first non-specific cholinesterase and then acetylcholinesterase in the same section. The different distributions of the two cholinesterases were confirmed with this method.With 8 Figures in the Text, of which 2 in ColourThis work has been supported by a research grant from the National Institute of Arthritis and Metabolic Disease, the U.S. Public Health Service (A-1725) and by a grant from Finland's Cultural Fund.     

Effect of pesticides on cholinesterase activity in the nervous elements of the heart in pregnant animals and fetuses]

Cholinestarase activity in the cardiac neural elements of pregnant animals and in their offspring was studied under the effect of chlororganic pesticide -- polychlorcamphen and phosphororganic pesticide -- valexone. The substances in question were demonstrated to produce variously pronounced unidirected changes in cholinesterase activity in neural structures that testifies to interconnection and dependence of the changes in the system mother -- fetus. The alterations observed in nonsynaptic (trophic) cholinesterase make it possible to speak about the disorder of cholinergic innervation and about the importance of acetilcholinesterase trophic function as a regulator of cellular processes, an inhibitory index of differentiation during embryogenesis.