Non-specific Cholinesterase Research Articles

The Pharmacology of Physostigmine

The Pharmacology of Physostigmine

Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CSF of rats with forebrain cholinergic system lesions.

Abnormal deposition and accumulation of Alzheimer's amyloid beta-protein (A beta) and degeneration of forebrain cholinergic neurons are among the principal features of Alzheimer's disease. Studies in rat model systems have shown that forebrain cholinergic deficits are accompanied by induction of cortical beta-amyloid precursor protein (beta-APP) mRNAs and increased levels of secreted beta-APP in the CSF. The studies reported here determined whether the CSF levels of secreted beta-APP could be altered pharmacologically. In different experiments, rats with lesions of the forebrain cholinergic system received injections of vehicle, a muscarinic receptor antagonist scopolamine, or one of two cholinesterase inhibitors - diisopropyl phosphorofluoridate (DFP) or phenserine. Scopolamine was administered to determine whether the levels of beta-APP in the CSF could be increased by anticholinergic agents. The cholinesterase inhibitors were administered to determine whether the forebrain cholinergic system lesion-induced increases in CSF beta-APP could be reduced by cholinergic augmentation. Scopolamine administration led to a significant increase in the CSF levels of secreted beta-APP in sham-lesioned rats. Phenserine, a novel, reversible acetyl-selective cholinesterase inhibitor, significantly decreased the levels of secreted beta-APP in the CSF of forebrain cholinergic system-lesioned rats whereas DFP, a relatively non-specific cholinesterase inhibitor, failed to affect CSF levels of secreted beta-APP. These results suggest that the levels of secreted beta-APP in the CSF can be pharmacologically modulated but that this modulation is dependent upon the status of the forebrain cholinergic system and the pharmacological properties of the drugs used to influence it.

Degeneration and regeneration of cutaneous sensory nerve formations.

Auxiliary structures of the cutaneous sensory nerve formations (SNF) are dependent on sensory innervation during their critical period of development. Denervation of mature cutaneous corpuscles results in survival of the terminal Schwann cells and the capsular structures which are probably responsible for successful reinnervation of the cutaneous SNF. In addition, the basal lamina tubes of Schwann cells are connected with the terminal Schwann cells and play an important role in the guidance of regrowing axons to their original targets. Long-lasting denervation causes atrophic changes of the terminal Schwann cells and alterations of their molecular equipment. These atrophic changes in the terminal Schwann cells may be responsible for erroneous reinnervation of cutaneous SNF. A population of the cutaneous Merkel cells surviving denervation may also serve as targets for regrowing sensory axons. The basal laminae of terminal Schwann cells are produced under control of the sensory terminals during maturation of cutaneous SNF. In adult animals, the basal laminae are capable of stimulating differentiation of migrated Schwann cells to the terminal Schwann cells without the presence of the sensory terminals. Nonspecific cholinesterase (nChE) is secreted by the terminal Schwann cells and is attached to their extracellular matrix. The synthesis of these molecules in adult animals is not influenced by the sensory terminals. However, the presence of nChE molecules is associated with living terminal Schwann cells. Fetal orthotopically grafted dorsal root ganglion (DRG) neurons have the ability to reinnervate cutaneous SNF of adult hosts. When cutaneous areas are denervated, axons from adjacent sensory nerves may extend collateral branches into this area. The capacity for such extension is dependent on: (1) type of sensory nerve ending, C and A delta fibers having significantly greater capacity than sensory axons of larger caliber; (2) age of the animal, immature animals generally showing a greater capacity for collateral sprouting; (3) the state of the adjacent axons, those already in a growth mode being more capable than "resting" ones; and (4) the regional and mechanical conditions at the site of denervation, hindpaw skin being much less extensively reinnervated by collateral fibers than that of the trunk.

Degeneration and regeneration of cutaneous sensory nerve formations

Auxiliary structures of the cutaneous sensory nerve formations (SNF) are dependent on sensory innervation during their critical period of development. Denervation of mature cutaneous corpuscles results in survival of the terminal Schwann cells and the capsular structures which are probably responsible for successful reinnervation of the cutaneous SNF. In addition, the basal lamina tubes of Schwann cells are connected with the terminal Schwann cells and play an important role in the guidance of regrowing axons to their original targets. Long-lasting denervation causes atrophic changes of the terminal Schwann cells and alterations of their molecular equipment. These atrophic changes in the terminal Schwann cells may be responsible for erroneous reinnervation of cutaneous SNF. A population of the cutaneous Merkel cells surviving denervation may also serve as targets for regrowing sensory axons. The basal laminae of terminal Schwann cells are produced under control of the sensory terminals during maturation of cutaneous SNF. In adult animals, the basal laminae are capable of stimulating differentiation of migrated Schwann cells to the terminal Schwann cells without the presence of the sensory terminals. Nonspecific cholinesterase (nChE) is secreted by the terminal Schwann cells and is attached to their extracellular matrix. The synthesis of these molecules in adult animals is not influenced by the sensory terminals. However, the presence of nChE molecules is associated with living terminal Schwann cells. Fetal orthotopically grafted dorsal root ganglion (DRG) neurons have the ability to reinnervate cutaneous SNF of adult hosts. When cutaneous areas are denervated, axons from adjacent sensory nerves may extend collateral branches into this area. The capacity for such extension is dependent on: (1) type of sensory nerve ending, C and Aδ fibers having significantly greater capacity than sensory axons of larger caliber; (2) age of the animal, immature animals generally showing a greater capacity for collateral sprouting; (3) the state of the adjacent axons, those already in a growth mode being more capable than “resting” ones; and (4) the regional and mechanical conditions at the site of denervation, hindpaw skin being much less extensively reinnervated by collateral fibers than that of the trunk. © 1996 Wiley-Liss, Inc.

Enzyme histochemistry of cutaneous sensory nerve formations.

The cutaneous sensory nerve formations belong to the structures which are studied intensely by the enzyme activity histochemistry since the early history of this technique. The histochemical localization of the activities of nonspecific cholinesterase, alkaline phosphatases, acid phosphatase, adenosine tri- and diphosphatases, adenylate cyclase, and dipeptidylpeptidase-IV in the cutaneous sensory nerve formations, mainly sensory corpuscles, is reviewed. The histochemical approach has brought new knowledge of both morphological building of these unique structures and their biochemical constituents. Taken together, the present results of enzyme histochemistry provide insight into the function of enzymes, and disclose new relationships between the sensory terminals and auxiliary structures in the cutaneous sensory nerve formations.

Age-Related Changes in Meissner Corpuscles in the Mouse Palate: A Histochemical and Ultrastructural Study.

Meissner corpuscles in the palatine mucosae of ddY-mice of various ages were studied histochemically and electron microscopically. As the age of the animals advanced, regressive Meissner corpuscles with lessened or depleted activity of non-specific cholinesterase (ChE) increased in number. Immunohistochemistry for Na+/K(+)-ATPase showed that the complex ramification of axon terminals in Meissner corpuscles attained at maturation gradually changed to a sparse distribution as the animals aged. Only a few putative rudiments of Meissner corpuscles, which were completely devoid of nerve terminals, were encountered. Electron microscopy showed that the regressive Meissner corpuscles contained atrophic axons and lamellae in a disordered arrangement, although the severity of the atrophy differed depending on the degree of the regression. Furthermore, the Meissner corpuscles were found to progressively accumulate fibrous long-spacing fibers having a periodicity of 160 nm and unique spherical corpuscles 150-300 nm in diameter in their intercellular spaces. From these results we concluded that: 1) the atrophy of the axon and lamellae proceeds almost simultaneously during the age-related regression of Meissner corpuscles; and 2) not only the atrophy of the axon and the lamellar cell but microenvironmental changes such as the accumulation of intercellular substances is also involved in the regression of these corpuscles.

Phorbol ester stimulates acetylcholine synthesis in cultured endothelial cells isolated from porcine cerebral microvessels

Acetylcholine (ACh) is one of the factor which induces vasodilation through the release of endothelium-derived relaxing factor. The aim of this study was to clarify whether endothelial cells can synthesize ACh and the types of substance which regulate the synthesis of ACh in endothelial cells. We determined the ACh content of endothelial cells isolated from porcine cerebral microvessels and of the culture medium. ACh was detected in the medium after 12 h incubation in the presence of diisopropylfluorophosphate, a non-specific cholinesterase inhibitor, and increased linearly up to 24 h. Phorbol 12-myristate 13-acetate (PMA, 10 −7 M) increased the ACh content of the medium in a dose-dependent manner. The effect of PMA was most apparent between 12 and 24 h after treatment, and was inhibited by cycloheximide. Calphostin C, a specific inhibitor of protein kinase C (PKC), did not inhibit the effect of PMA. Dioctanoyl glycerol, a specific activator of PKC, did not increase the intracellular ACh content or the amount released into the culture medium. ACh synthesis was not inhibited by bromoacetylcholine, a specific inhibitor of choline acetyltransferase (ChAT). PMA treatment did not affect the specific activity of ACh synthesis in endothelial cells. These data show that endothelial cells are able to synthesize ACh, and that ACh synthesis is up-regulated by PMA through the PKC independent mechanism via protein induction. The enzyme which synthesizes ACh in endothelial cells is not ChAT. The increase in ACh synthesis induced by PMA may not be due to induction of the ACh synthetic enzyme.

Butyrylcholinesterase from intestinal epithelial cells of quail, chick and duck: A comparative study during development

1. 1. Epithelial cells of avian intestine express non-specific cholinesterase (butyrylcholinesterase, BuChE) activity both in the embryo and adult animal. 2. 2. Quail, duck and chick exhibit increased BuChE activity during the late embryonic period followed by decreased activity. The minimum value is reached after hatching at day 1 in quail, day 4 in chick and day 6 in duck. 3. 3. The three species display marked sex-related differences mainly during the peri-hatching period. BuChE activity is higher in females than in males. 4. 4. The three globular forms, G 1, G 2 and G 4, are detected during the last days of embryonic development. After hatching, changes in the relative amounts of these forms are related to the disappearance of G 2 and the gradual decrease of G 4. In adult quail and duck, G 4 is not detected and BuChE activity corresponds only to the G 1 form. 5. 5. The changes in BuChE activity and distribution of the molecular forms occur in a similar manner in all three species although the embryonic periods differ notably, suggesting that hormonal factors secreted during this period and involved in the preparation of hatching may regulate BuChE activity.

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.

The nervous system of Fibricola seoulensis by acetylcholinesterase histochemistry

F. seoulensis were obtained from artificially infected albino rats at 3, 4, 5, 6, 7 days after infection. The worms and metacercariae were washed in physiological saline solution, and fixed with 10% neutral formalin. The acetylcholinesterase (AchE) stained by enzyme histochemistry using acetylthiocholine iodide as substrate. Eserine, iso-OMPA and BW284C51 were used as inhibitors of AchE. The nervous system consists of three pairs longitudinal nerve trunks interconnected with excretory plexus in posterior half, and pharynx and oral sucker in anterior half of metacercariae and adults. The longitudinal nerve trunks are interconnected with transverse commissures and numerous circular commissures. Considerable numbers of circular commissures are interconnected with longitudinal nerve trunks lying on the surface of the worms. At each stage of juvenile worms, AchE and nonspecific cholinesterase activities were observed in the oral sucker, ventral sucker, pharynx and nerve system. Isozymes of AchE in F. seoulensis were separated into the two bands, 69 kDa and 132 kDa. The major band was 69 kDa.

Origin of cells in contact with the growth cones of embryonal peripheral nerves and histochemical detection of nonspecific cholinesterase activity in quail-chick and chick-quail chimeras.

The types of cells contacted by growth cones were examined in chick-quail and vice versa chimeras. Simultaneously, the presence of nonspecific cholinesterase (nChE) was monitored in developing peripheral nerves of these embryos. In all the chimeras studied, the growth cones were in contact with both Schwann cell progenitors and mesenchymal cells of the limb bud. This observation implies that the growth cones may obtained guidance cues directly from the mesenchymal cells of the limb bud. Schwann cell progenitors as well as mesenchymal cells in contact with growth cones were capable to produce nChE molecules. The reaction product indicating nChE activity was localized on the outer surface of the plasma membrane of both types of cells in contact with growth cones. However, mesenchymal cells forming the "primitive perineurium" were devoid of the reaction product on their plasma membrane. We conclude that with exception of classic adhesive molecules the process of axonal guidance can be mediated by nChE molecules.

Migration of Schwann cells from the distal stump of the sciatic nerve 1 week after transection: the effects of insulin and cytosine arabinoside.

We have examined the migratory capacity of Schwann cells from the distal stump of a 1-week transected sciatic nerve of adult rat for a distance of 10 mm. The distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon) soaked in phosphate-buffered saline (control chambers), cytosine arabinoside (Ara-C) (0.05 mM), or insulin (40 U/ml). Migrating Schwann cells were distinguished from fibroblasts by the presence of non-specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP). The cells of distal stumps including Schwann cells accepted Gelaspon as a suitable adhesive substratum. In the chambers filled with Gelaspon soaked in phosphate-buffered saline alone Schwann cells were outnumbered by fibroblasts. The addition of Ara-C resulted in greater numbers of Schwann cells, which migrate longer distances into the chambers. The application of insulin enhanced Schwann cell migration as well. These morphologic observations were further supported by biochemical measurements of nChE activity. The results suggest an influence on Schwann cell migration by fibroblasts of connective tissue sheaths and a stimulation of Schwann cell migration by insulin.

A histochemical study of the activity of nonspecific cholinesterase in C6 glioma cells in culture.

The activity of nonspecific cholinesterase (nChE) was revealed histochemically in a small number of C6 glioma cells under conditions of spontaneous growth in culture. The nChE-positive cells (nChE+) first appeared in 4-day-old cultures. They occurred solitarily or in small groups with a cluster- or chain-like arrangement. The incidence of nChE+ cells increased with in vitro age and/or higher population densities of cultures but it never exceeded 3% of the whole population. These results indicate the necessity of histochemical as well as biochemical data on nChE activity in glioma cells both in situ and in culture for the discovery of the heterogeneous distribution of this enzyme. db-cAMP induced a decrease in the proportion of nChE+ cells to total number of cells. These changes suggest that the original C6 glioma cell seed stock contains a small subclone of less differentiated cells exhibiting nChE activity. The greater number of nChE+ cells in older cultures is probably due to recovery and multiplication of initially scarce subclone cells. C6 glioma cells in culture form a suitable model for investigation of changes in nChE activity of glial cells provided that a histochemical method of detection is applied.

Development of esterase activities in the chicken before and after hatching

The embryonic chick has long been a model for developmental biology and has often been recommended as a model system in developmental toxicology. More recently, several investigators have shown that the chick embryo also provides a good model for identifying the neurotoxic effects of environmental pollutants, especially cholinesterase-inhibiting pesticides. Although numerous studies detail the structural development of chick embryos, few describe embryonic levels of enzyme synthesis and their changes during development. In this study, the development of esterase activity in chick embryos was measured from day 9 of incubation until 46 days after hatching. Brain acetylcholinesterase (AChE) activity was detected on day 9 of incubation at a concentration of 0.364 μmoles/min/g tissue. An increase between AChE activity and age of the embryos was observed. In the liver, the nonspecific cholinesterases (ChE) and carboxylesterase activities during incubation were not different from activities after the chicks had hatched. Plasma ChE and carboxylesterase activities did not change with age after hatching. Brain neuropathy target esterase (NTE) activity was not detected on day 9 of incubation and was extremely low (6.12 nmoles/15 min/mg protein) the next day, but increased rapidly with increasing age. This study demonstrates that chick embryos have developed esterase activities in the brain and liver by day 10 of incubation and again confirms that the insensitivity of chick embryos and young chicks to organophosphorus ester-induced delayed neurotoxicity is not due to absence of NTE. In addition, the results provide baseline data for evaluating the response of embryonic and immature chicks to neurotoxicants and teratogens.

Cholinesterase activity in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors

A nonspecific cholinesterase activity was demonstrated in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors at the light and electron microscopic levels. The terminal Schwann cells are ultrastructurally characterized by a well-developed Golgi apparatus and rough endoplasmic reticulum. The cells in this study were positive for nonspecific cholinesterase, whereas ordinary Schwann cells associated with more proximal nerve fibers reacted negatively. The reaction products were densely deposited in the cisternae of the rough endoplasmic reticulum and along the nuclear envelop. A moderately intense labeling was found in the cytoplasmic extensions, in which the reaction products gathered in caveolae and vesicles. These findings indicate that nonspecific cholinesterase is a useful marker to distinguish terminal Schwann cells from ordinary Schwann cells and that the enzyme may be synthesized in the rough endoplasmic reticulum and conveyed toward the axon terminals. Since this enzyme has been known to be shared by the inner bulb of Pacinian corpuscles and the lamellar cells of Meissner's corpuscles, its possible involvement in mechanoreceptive functions in these specialized Schwann cells deserves further investigation.

Synthesis and release of acetylcholine by cultured bovine arterial endothelial cells

Using cultured endothelial cells prepared from bovine carotid artery, and a specific radioimmunoassay for acetylcholine (ACh), the synthesis and release of ACh by vascular endothelial cells were investigated directly. ACh content in the culture medium after 24 h of incubation in the presence of isoflurophate, a nonspecific cholinesterase inhibitor, was about 16 times higher than that in endothelial cells, indicating that ACh synthesized inside the cells was released rapidly. The presence of ACh in the culture medium was further confirmed qualitatively using high-performance liquid chromatography with an electrocapture detection. These results represent the first direct evidence that endothelial cells can synthesize and release ACh, suggesting the possibility that ACh acts not only as a neurotransmitter but also as an autacoid under certain conditions.

Cholinesterase Activity in Sensory-Nerve Endings, Capillaries and Motor End Plates of the Facial Skin of the Brush-Tailed Possum (<i>Trichosurus vulpecula</i>)

The facial skin of two adult and one 50-day-old pouch animal of the marsupial mammal Trichosurus vulpecula was removed after the animals has been suitably anesthetized and perfused for electron microscopy. Small blocks of tissue (1 x 0.5 mm) were cut and incubated in acetylthiocholine iodide substrate for cholinesterase studies. The blocks were then subsequently postfixed in osmium tetroxide. Thin sections were cut and stained with lead acetate. Specific cholinesterase was found within the nerves of both the adult and the 50-day-old pouch animal, and in the motor end plates. Nonspecific cholinesterase was present in pinocytotic vesicles and interlamellar spaces of terminal Schwann cells associated with nerve end organs in the adult, and in the same areas in Schwann cells of nonmyelinated nerves in the pouch animal. It was also present in the pinocytotic vesicles of the capillary endothelium.

Non-specific cholinesterase activity of the developing peripheral nerves and its possible function in cells in intimate contact with growing axons of chick embryo

The results presented here demonstrate non-specific cholinesterase (nChE) activity in the developing peripheral nerves of chick embryos at stages 25–26 according to Hamburger and Hamilton (1951, J. Morphol. 88, 49–92). Under the light microscope the use of simultaneous staining for nChE activity and silver proteinate impregnation revealed the axons to be surrounded by cells exhibiting nChE activity in the main nerve trunks and in the growing tips of nerves. Nerve branches arising from the main nerve trunks contained cells with positive reaction for nChE activity, too. Electron-dense particles of the reaction product indicating nChE activity were found in the rough endoplasmic reticulum and in the perinuclear envelope of cells in close contact with growing nerve fibers and their growth cones. The same distribution of nChE activity was found in cells which were located near to nerve fasciculi but without direct contact with axons. Surprisingly, the cells in close contact with axons and their growth cones exhibited the end product of nChE activity in the outer part of their plasma membrane. The cells enveloping axons within the nerve trunks were apparently Schwann cells, while those around the growth cones at nerve tips could be identified as Schwann cells and/or mesenchymal cells of the hindlimb. The nChE reaction product was also detected in the axolemma of nerve fibers and their growth cones. The distribution of nChE activity in the developing peripheral nerves of chick embryos suggests that these molecules may influence the process of axonal elongation and locomotion. Several possible mechanisms of nChE action on growing axons can be presumed: 1.(i) intracellular Ca2+ level regulation.2.(ii) providing an adhesive substrate.3.(iii) butyrate production affecting the cell metabolism and the distribution of neurotubules and neurofilaments. It is also assumed that nChE molecules are involved in the interactions of nerve fibers with Schwann cells and/or mesenchymal cells as well as in interneuronal interactions.

Recovery of non-specific cholinesterase activityin sensory corpuscles of mouse toe skin after irreversible inhibition of this enzyme and cold injury

Mouse digital corpuscles, located in the dermal papillae of toe pad skin, consist of the sensory axon terminals enveloped by the cytoplasmic processes of Schwann-derived cells forming the so-called inner core. The inner core cells are capable to synthetize nCHE molecules which are released into the interlamellar spaces filled by the basal lamina, collagenous microfibrils, and amorphous matrix. In the present study, the histochemical detection of the nCHE activity was investigated in the sensory corpuscles after sciatic and saphenous nerve transections and subsequent application of irreversible nCHE inhibitor (iso-OMPA) or cryo-treatment of toe pad skin. The recovery of the nCHE reaction product in both intact and denervated corpuscles revealed the resynthesis of the nCHE molecules by the inner core cells without assistance of sensory terminals, as well. The cellular constituents of corpuscles were degraded while extracellular matrix appeared to be undamaged after freezing injury. The molecules of nCHE attached to the extracellular matrix components disappeared in coincidence with the disintegration of Schwann-derived cells. After about 5 d of survival, the Schwann cells exhibiting the nCHE reactivity migrated through the basal lamina tubes as guidance of regrowing axons or alone. After 7 d from the treatment, immature Schwann cells marked by the nCHE reaction product occupied the scaffolds of old damaged sensory corpuscles. During further days of surviving, the Schwann cells entering the extracellular matrix of degraded corpuscles were differentiated to the inner core cells. The re-differentiation of the Schwann cells into the inner core cells was observed not only in the presence but also in the absence of sensory terminals. These findings suggest certain trophic independence of inner core cells upon sensory terminals in the sensory corpuscles of adult animals.

Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties.

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.