The mean miniature end-plate potential (m.e.p.p.) frequency and the acetylcholinesterase (AChE) activity have been examined in the rat diaphragm, in relation to the time of Li+ action. Substitution of LiCI for NaCI caused increase in m.e.p.p. frequency, the final level of which depended on Li + concentration in the bath solution. Incubation of diaphragm homogenate with NaCI caused initially a slight increase of AChE activity, reaching a plateau after 20 min. Substitution of LiCI for NaCI produced an increase in AChE activity to a maximum, followed by a decrease to a minimum. The activity consequently returned to a steady level. During complete substitution of LiCI for NaCI, the maximum value of AChE activity coincided with the maximum m.e.p.p. frequency (Finax) induced by Li +; the minimum value correlated with the precise instant that the frequency reached a plateau. The addition of prostigmine caused: a) blockade of AChE activity, and b) prostigmine concentration-dependent decrease of Fmax and/or disappearance of m.e.p.p.s from neuromuscular junctions treated with Li+. These results are discussed in terms of correlation between changes of AChE activity induced by Li+ and variations of spontaneous quantal release of the transmitter from nerve terminals of the rat diaphragm treated with Li + . It is almost 40 years since CADE (1949) rediscovered the therapeutic properties of Li in the treatment of manic excitement, and Li salts are now firmly established in modern psychiatric treatment. Since OVERTON (1902) first showed that muscle retained its excitability in Li solution, there has been much interest devoted to the effect of Li + on the electrical properties of excitable cells. It was found that the action potential is little affected when the Na+ of the external solution is replaced by Li + (HODGKIN and KATZ, 1949;HUXLEY and STAMPFLI, 1951) and the Na +
3 ) respectively, after preincubation with Phe. A C h E activity in brain homogenate or in pure eel E.electricus enzyme showed a decrease, which reached up to 18% with concentrations of 0 .9 -1 2 .1 m M . Brain homogenate N a+,K +-A TPase activity showed an increase 1 6 -6 5 % with 0 .2 4 -0 .9 mM of Phe, while an activity increase of 6 0 -6 5 % appeared with 0 .9 -1 2 .1 mM of Phe. Pure en zyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations, while it was increased by low concentrations. The above results suggest: a) A direct effect of Phe on A C h E , b) A direct effect of low Phe concentrations and an indirect effect of high ones on Na+,K +-ATPase. IntroductionPhenylketonuria (PK U ) is a group of recessively inherited metabolic disorders, in which the conver sion of phenylalanine (Phe) to tyrosine is im paired. This metabolic disorder is due to an au tosomal recessive gene that codes for a type of phenylalanine hydroxylase that has reduced enzy matic activity, resulting in abnormally high levels of Phe in body fluids (Kupfermann, 1991). An ex cessive increase of blood Phe results in mental re tardation (IQ <60) (Missiou-Tsagaraki et al., 1988), seizures and other neurophysiological and psycho logical dysfunctions (Behbenhani and Langenbeck, 1982). High Phe concentrations (0 .3 -1 . 8 mM) may occur in sick humans, and are harm ful especially during the first year of life, while this "damage" can be prevented with an appropriate dietary control (Missiou-Tsagaraki et al., 1988).The rapid and precise communication between neurons, necessary for the performance of nervous system functions, is made possible by two signaling mechanisms: excitability and synaptic trans mission.Neural excitability was found to be influenced by Phe in rat brain (Iarosh et al, 1987) while ex perimental hyperphenylalaninemia in 3 -1 7 dayold rats leads to reduced myelinogenesis (Burri et a l, 1990), which could result in a decrease of the axonal conduction velocity. Reduced myelinogen esis is consistent with the observation which showed that high Phe concentrations in the body fluids inhibit brain protein synthesis by inhibition of the transport of amino acids across the bloodbrain barrier (Oldendorf, 1973; Antonas and Coulson, 1975) or by direct interference with the pro tein synthesis apparatus (Taub and Johnson, 1975; Huges and Johnson, 1977).Alterations in synaptic transmission are also im plicated in brain dysfunctions in PKU and several experimental data suggest that the principal cause for the brain dysfunction is the impairment in the neurotransmitter amine synthesis (Blau, 1979). biosynthetic precursors for the neurotransmitters serotonin, dopamine and norepinephrine (N E), the aromatic amino acids uptake into the brain can modify their convertion to neurotransmitters and thereby can modify their release from neurons and influence brain functions (Fernstrom, 1994). Moreover high Phe concentrations induce changes of brain electrical function, which may be medi ated in part through inhibitio...
The effect of different L-phenylalanine (Phe) concentrations (0.24-12.1 mᴍ) , on acetylcholinesterase (AChE) and Na+,K+-ATPase activities of diaphragm homogenates from 21-day old rats and pure enzymes, was investigated at 37 °C. AChE and N a+,K+-ATPase activities were determined after preincubation with Phe. AChE activity in diaphragm homogenate or in pure eel E. electricus enzyme showed a decrease, which reached a maximum of 18% with Phe concentrations of 0.9-12.1 mᴍ. However lower Phe concentrations (0.24 mᴍ) increased the enzyme activity (by approximately 22%), only in the diaphragm homogenate. Diaphragm-associated Na+,K+-ATPase activity showed a progressive and concentration-depen dent decrease, by about 30-35% in the presence of high Phe concentrations. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations (>0.48 m M) , while it was increased by low concentrations. The above results suggest; a) A direct inactivating effect of high Phe concentrations on AChE and an indirect activating effect induced by low concentrations, b) A direct activating effect of low Phe concentrations and an indirect inactivating effect of high ones on Na+,K+-ATPase. c) The combination of high Phe concentrations effects on AChE and Na+,K+-ATPase could influence the levels of the diaphragm synaptic ACh.
Acetylcholinesterase (AChE) activity has been determined using homogenized rat diaphragm and soluble AChE from the eel Electrophorus electricus, using as a substrate different amounts of acetylthiocholine in the presence or absence of 115 mM NaCl or LiCl. With LiCl the KM values derived from Lineweaver-Burk plots are found to be 470-650 and 1045-1425 microM without cations or with NaCl. The cooperativity of the enzyme is increased when cations are added to the homogenate, as demonstrated by changes of the Hill coefficient. With soluble AChE, only Li+ is able to produce this effect. Preincubation of the soluble enzyme at low pH (5.5) and a change to a higher value (8.7-9.4) causes a decrease of the Hill coefficient with Li+ only; this effect is not detected using the homogenate. Our results suggest the following. (i) Li+ may neutralize negative charges of AChE more successfully than does Na+, resulting in higher activity, stabilization, and cooperativity of the enzyme. (ii) The KM values calculated at high substrate concentrations (greater than 200 microM) indicate that the substrate affinity of AChE can be increased only by Li+ binding on the enzyme. (iii) Changes in pH can modulate the cooperativity and may denature allosteric sites on the enzyme that bind Li+. (iv) Membrane, cations and (or) cellular factor(s) may regulate the cooperativity and substrate affinity of AChE, when they have been affected by pH changes.
Effets de la substitution de lithium au sodium dans le milieu de survie sur la libération spontanée de transmetteur par les terminaisons motrices du diaphragme isolé de Rat.
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