The interaction of quaternary reversible acetylcholinesterase inhibitors with the nicotinic receptor.

Acetylcholinesterase inhibitors (AChEIs) are used in the treatment of myasthenia gravis (MG). We investigated the effects of AChEIs on peripheral nicotinic receptors (nAChR), which play a crucial role in the treatment of MG symptoms. The positive modulation of those receptors by AChE inhibitors could have an added value to the anti-AChE activity and might be useful in the therapy of MG. Furthermore, to estimate the potential drawbacks of the compounds, cytotoxicity has been assessed on various cell lines. The whole-cell mode of the patch-clamp method was employed. The experiments were performed on medulloblastoma/rhabdomyosarcoma cell line TE671 expressing human embryonic muscle-like receptor with subunits alpha2betagammadelta. The effect of the compounds on cell viability was measured by standard MTT assay (Sigma Aldrich) on ACHN (renal cell adenocarcinoma), HeLa (immortal cell line derived from a cervical carcinoma), HEPG2 (hepatocellular carcinoma) and BJ (skin fibroblasts) cell lines. No positive modulation by the tested AChE inhibitors was observed. Moreover, the compounds exhibited antagonistic activity on the peripheral nAChR. Standard drugs used in MG treatment were shown to be less potent inhibitors of muscle-type nAChR than the newly synthesized compounds. The new compounds showed very little effect on cell viability, and toxicities were comparable to standards. Newly synthesized AChEIs inhibited peripheral nAChR. Furthermore, the inhibition was higher than that of standards used for the treatment of MG. They could be used for the study of nAChR function, thanks to their high antagonizing potency and fast recovery of receptor activity after their removal. However, since no positive modulation was observed, the new compounds do not seem to be promising candidates for MG treatment, even though their cytotoxic effect was relatively low.

Chloride cotransport in the membrane of earthworm body wall muscles.

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).

Specific binding to plasma membrane is the first step in the uptake of non-transferrin iron by cultured cells.

We studied transport of non-transferrin iron into HeLa cells adapted for growth in defined medium, containing either 5 micrograms/ml of iron-saturated transferrin (HeLa/Tf cells) or 5 microM ferric citrate (HeLa/Fe5 cells) as a source of iron. Employing 55Fe-ferric citrate, iron uptake by intact cells was compared with iron binding to isolated membranes. Uptake characteristics of both HeLa/Tf and HeLa/Fe5 cells seemed to be similar: Km = 14 microM and Vmax = 135 pmol Fe/min/10(5) cells for HeLa/Tf, Km = 22 microM and Vmax = 165 pmol Fe/min/10(5) cells for HeLa/Fe5. Increasing concentrations (0.3-1.2 microM) of 55Fe-ferric citrate, producing levels of free 55Fe which were independent of total Fe under the experimental conditions used, led to increased binding of 55Fe for both HeLa/Tf and HeLa/Fe5 cells (1.08-8.03 nmol Fe/h/10(5) cells). This corresponds with the suggestion that iron was bound in the form of ferric citrate rather than in the form of free iron. Dissociation constants of Fe binding, KD = 0.61 microM for HeLa/Tf and KD = 1.53 microM for HeLa/Fe5, were obtained from competition experiments. We conclude that specific binding sites for ferric citrate are constitutively expressed in plasma membrane and that their expression does not require the induction by the presence of ferric citrate. The uptake of non-transferrin iron is realized in at least two steps. The first step is iron binding to the specific binding sites in plasma membrane. The binding does not represent a limiting step of the uptake.

Electrophysiological characterization of GABAA receptors in anterior pituitary cells of newborn rats.

The gamma-aminobutyric acid (GABA)-ergic communication between the CNS and the anterior pituitary gland has been documented in numerous histochemical and biochemical studies but electrophysiological studies characterizing the GABAA receptor in the anterior pituitary are still lacking. In the present report we studied the GABA-induced current responses in cultured cells from the anterior pituitary gland of 6- to 10-day-old rats using the patch-clamp technique in the whole cell configuration. Fast application of GABA (100 microM) induced membrane currents in 90% of cells in 2-day-old cultures. The EC50 for GABA was 22.9 microM and the Hill coefficient was 1.8. The responses to GABA (10 microM) were inhibited by bicuculline (2 microM) to 14%, by picrotoxin (5 microM) to 21% and by zinc (10 microM) to 33%. Inhibition to 56% was observed with 6 microM strychnine. The GABA responses were sensitive to diazepam and pentobarbital. Half-maximal potentiation of responses to GABA (10 microM) was found with 1.0 microM diazepam and with 14.4 microM pentobarbital. The maximal potentiation of GABA responses was 222% for diazepam and 195% for pentobarbital. Pentobarbital (100 microM) did not induce any response in anterior pituitary cells in the absence of GABA. The application of GABA at concentrations 10 microM or higher, induced membrane currents that desensitized. Desensitization proceeded as a biexponential process with estimated fast and slow time constants which decreased with concentration. The responses to GABA (300 microM) desensitized to 93% with time constants of 1.4 and 5.3 s. Half-maximal desensitization was found with 13.4 microM GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of non-quantal release of acetylcholine in regulation of postsynaptic membrane electrogenesis.

In mammalian nerve-muscle preparations treated with an anticholinesterase, the acetylcholine (ACh) released non-quantally (NQR) reaches the postsynaptic receptors and causes a small depolarization of the membrane potential at the endplate region of the muscle fibres. Increase in quantal release potentiates the NQR and vice versa, the amplitude and the kinetic parameters of quantal miniature endplate currents (MEPCs) change during manipulation of NQR, indicating direct interaction between both types of release. Repetitive binding of ACh to postsynaptic receptors which prolongs the time course of MEPCs in anti-cholinesterase-treated endplates leads within 1-2 h to progressive desensitization in the presence of non-quantal release and to the subsequent shortening of the quantal responses. We have also investigated the effect of procedures known to modulate non-quantal acetylcholine release, on the small, but obvious, difference in the resting membrane potential between the endplate zone and other areas of the mouse muscle fibre. The resting membrane potential at the endplate zone with intact cholinesterase is more negative (by 2-4 mV) than in the endplate-free area. The experiments were performed to test the hypothesis that the hyperpolarization is caused by an electrogenic Na(+)-K+ pump operating during the action of ACh released in non-quantal form. Observations in favour of this idea are that both short-term denervation (which eliminates non-quantal but not quantal release) and ouabain abolish the local synaptic hyperpolarization and that subsequent application of low doses of ACh restores it. It follows, therefore, that the hyperpolarization is probably caused by a small but continuous ACh leakage from the nerve terminal.