Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Abstract

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.

PKC stimulation increases expression of Cholinesterases in R28 cell line

“…DOG is a diacyl glycerol analog of the classical stimulator of calcium-dependent PKC-α signaling. Previously, the interaction between ChE and PKC-α is shown [7,[8][9][10][11][12][13][14]. In this study, we stimulated embryonic cell line R28 with DOG under normal cell conditions as well as knocking BChE down.…”

“…AChE contains a Ca 2+ binding motif, called an EF hand, and BChE can be activated through Ca 2+ in a concentration-dependent manner [10]. ChEs also regulate voltage-dependent Ca 2+ currents and neurite growth [11,12]. In addition, Ca 2+ reactivates inhibited BChE, signifying an important link between Ca 2+ and functionally active ChE proteins [13].…”

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

“…There is mounting evidence that ChEs may be involved in functions distinct from their catalytic activity. During development of the nervous system of vertebrates, expression of ChEs is associated with periods of enhanced neurite outgrowth Layer et al, 1993), suggesting that they might serve as neuronal growth factors or cell-adhesion molecules. It has been speculated that the site responsible for the non-catalytic interaction of ChEs is localized close to the entrance of the active-site gorge, perhaps overlapping with what has been termed the 'peripheral' anionic site (PAS) of AChE, a possible locus of regulation of enzymatic activity Eichler et al, 1994;Barak et al, 1995), which is located in the region of the 'annular' electrostatic motif.…”

Biochemical Markers for Exposure to Low Doses of Organophosphorus Insecticides

“…Inhibition of AChE activity in neuronal cultures has no effect on neurite outgrowth if the inhibitor binds at the bottom of the active site gorge, for example when the inhibitor is diisopropylfluorophosphate [3,30]. However, an inhibitor that binds near the surface of the AChE protein, at the peripheral site, inhibits neunte outgrowth [3,15,23,28,32]. These results as well as the high sequence homology of AChE and cell adhesion proteins, have led to the hypothesis that the neuritogenic ability of AChE is mediated through a cell adhesion mechanism.…”

“…Overstimulation of these receptors may lead to respiratory failure and death. AChE has been proposed to have a second function independent of its catalytic activity [1][2][3][4]9,11,[13][14][15][16]23,25,26,28,[30][31][32], possibly serving as a cell adhesion factor in morphogenesis…”