Pregnane neurosteroids exert opposite effects on GABA and glycine-induced chloride current in isolated rat neurons.

The ability of endogenous neurosteroids (NSs) with pregnane skeleton modified at positions C-3 and C-5 to modulate the functional activity of inhibitory glycine receptors (GlyR) and ionotropic É£-aminobutyric acid receptors (GABAA R) was estimated. The glycine and GABA-induced chloride current (IGly and IGABA ) were measured in isolated pyramidal neurons of the rat hippocampus and in isolated rat cerebellar Purkinje cells, respectively. Our experiments demonstrated that pregnane NSs affected IGABA and IGly in a different manner. At low concentrations (up to 5 µM), tested pregnane NSs increased or did not change the peak amplitude of the IGABA , but reduced the IGly by decreasing the peak amplitude and/or accelerating desensitization. Namely, allopregnanolone (ALLO), epipregnanolone (EPI), pregnanolone (PA), pregnanolone sulfate (PAS) and 5ß-dihydroprogesterone (5ß-DHP) enhanced the IGABA in Purkinje cells. Dose-response curves plotted in the concentration range from 1 nM to 100 µM were smooth for EPI and 5ß-DHP, but bell-shaped for ALLO, PA and PAS. The peak amplitude of the IGly was reduced by PA, PAS, and 5α- and 5ß-DHP. In contrast, ALLO, ISO and EPI did not modulate it. Dose-response curves for the inhibition of the IGly peak amplitude were smooth for all active compounds. All NSs accelerated desensitization of the IGly . The dose-response relationship for this effect was smooth for ALLO, PA, PAS and 5ß-DHP, but it was U-shaped for EPI, 5α-DHP and ISO. These results, together with our previous results on NSs with androstane skeleton, offer comprehensive overview for understanding the mechanisms of effects of NSs on IGly and IGABA .

Factors promoting the release of picrotoxin from the trap in the GABA(A) receptor pore.

Picrotoxin (PTX), a convulsant of plant origin, has been used in many studies as research tool. PTX is the open channel blocker of the GABAA receptor (GABAAR). Being in the pore, PTX initiates transfer of the channel to the closed state and thus it falls into the "trap". The consequence of this PTX trapping is so-called aftereffect, i.e. continuation of the blockade of the GABA-induced chloride current (IGABA) after removal of PTX from the external solution. The present work shows that the positive allosteric modulators (PAMs) of the GABAA receptor, allopregnanolone (Allo) and zolpidem (Zolp) as well as a high concentration of GABA shortened the PTX aftereffect. Experiments were carried out on isolated Purkinje neurons of the rat cerebellum using the whole-cell patch-clamp method. IGABA was induced by applications of 5 µM GABA (EC30) for 1 s with 30 s intervals. 50 µM PTX completely blocked IGABA, and recovery upon PTX washout occurred with a time constant (τrec) of 20.2 min. 1 µM Allo reduced the blocking effect of PTX by 30% and accelerated the recovery of IGABA by almost 10 times (τrec = 2.4 min). 0.5 µM Zolp did not change the IGABA block in the presence of PTX but accelerated the recovery of IGABA by more than 3 times (τrec = 5.6 min). Increasing the GABA concentration to 20 µM did not change the blocking effect of PTX, but accelerated recovery by 6 times (τrec = 3.3 min). The mechanism of the shortening of the PTX aftereffect is presumably the expansion of the GABAAR pore in the presence of PAMs and a high concentration of the agonist and, as a consequence, the escape of PTX from the "trap". The work describes new pharmacological properties of Allo and Zolp.

Donepezil in low micromolar concentrations modulates voltage-gated potassium currents in pyramidal neurons of rat hippocampus.

Donepezil is a cholinesterase inhibitor widely used for the treatment of Alzheimer's disease. Voltage-gated K(+)-channels are discussed as possible targets for the drug, but the results obtained by different authors are contradictory. In the present study performed on pyramidal cells isolated from rat's hippocampus, we investigated the effect of donepezil on delayed rectifier K(+)-current (I(K(DR))) and transient outward K(+)-current (I(K(A))) using patch-clamp technique. The inhibitory effect of donepezil on I(K(DR)) was found in all the cells tested, but its strength varied in different cells. Two groups of neurons were differing in their sensitivity to donepezil: more sensitive (IC(50)=8.9 µM) and less sensitive (IC(50)=114.9 µM). The effect of the drug on I(K(DR)) was rapid, reversible and voltage-dependent, increasing with depolarization. Donepezil modulated I(K(A)) in two different ways: in some cells it suppressed the current with the IC(50) value of 23.4 µM, while in other cells it augmented the current with a bell-shaped dose-response curve. Maximal (about twofold) enhancement of I(K(A)) amplitude was caused by 10 µM donepezil. Augmentation of I(K(A)) increased with membrane depolarization. Our results show for the first time that voltage-dependent potassium channels in mammals' neurons are effectively modulated by low micromolar concentrations of donepezil.

Corticosteroids as Selective and Effective Modulators of Glycine Receptors.

The mechanism of the negative impact of corticosteroids on the induction and progress of mental illness remains unclear. In this work, we studied the effects of corticosteroids on the activity of neuronal glycine receptors (GlyR) and GABA-A receptors (GABAAR) by measuring the chloride current induced by the application of GABA (2 or 5 µM) to isolated cerebellar Purkinje cells (IGABA) and by the application of glycine (100 µM) to pyramidal neurons of the rat hippocampus (IGly). It was found that corticosterone, 5α-dihydrodeoxycorticosterone, allotetrahydrocorticosterone, cortisol, and 17α,21-dihydroxypregnenolone were able to accelerate the desensitization of the IGly at physiological concentrations (IC50 values varying from 0.39 to 0.72 µM). Next, cortisone, 11-deoxycortisol, 11-deoxycorticosterone, 5ß-dihydrodeoxycorticosterone, and tetrahydrocorticosterone accelerated the desensitization of IGly with IC50 values varying from 10.3 to 15.2 µM. Allotetrahydrocorticosterone and tetrahydrocorticosterone potentiated the IGABA albeit with high EC50 values (18-23 µM). The rest of the steroids had no effect on IGABA in the range of concentrations of 1-100 µM. Finally, our study has suggested a structural relationship of the 3ß-hydroxyl group/3-oxo group with the selective modulatory activity on GlyRs in contrast to the 3α-hydroxyl group that is pivotal for GABAARs. In summary, our results suggest that increased GlyR desensitization by corticosteroids may contribute to brain dysfunction under chronic stress and identify corticosteroids for further development as selective modulators of GlyRs.

Donepezil in a narrow concentration range augments control and impaired by beta-amyloid peptide hippocampal LTP in NMDAR-independent manner.

Acetylcholinesterase (AChE) inhibitor donepezil is widely used for the treatment of Alzheimer's disease (AD). The mechanisms of therapeutic effects of the drug are not well understood. The ability of donepezil to reverse a known pathogenic effect of ß-amyloid peptide (Abeta), namely, the impairment of hippocampal long-term potentiation (LTP), was not studied yet. The goal of the present study was to study the influence of donepezil in 0.1-10 µM concentrations on control and Abeta-impaired hippocampal LTP. Possible involvement of N-methyl-D: -aspartate receptors (NMDARs) into mechanisms of donepezil action was also studied. LTP of population spike (PS) was studied in the CA1 region of rat hippocampal slices. Change of LTP by donepezil treatment had a bell-shaped dose-response curve. The drug in concentrations of 0.1 and 1 µM did not change LTP while in concentration of 0.5 µM significantly increased it, and in concentration of 5 and 10 µM suppressed LTP partially or completely. Abeta (200 nM) markedly suppressed LTP. Addition of 0.1, 0.5 or 1 µM donepezil to Abeta solution caused a restoration of LTP. N-methyl-D: -aspartate (NMDA) currents were studied in acutely isolated pyramidal neurons from CA1 region of rat hippocampus. Neither Abeta, nor 0.5 µM donepezil were found to change NMDA currents, while 10 µM donepezil rapidly and reversibly depressed it. Results suggest that donepezil augments control and impaired by Abeta hippocampal LTP in NMDAR-independent manner. In general, our findings extend the understanding of mechanisms of therapeutic action of donepezil, especially at an early stage of AD, and maybe taken into account while considering the possibility of donepezil overdose.

Positive allosteric modulators of GABAA receptor restore chloride current from blockade by competitive antagonists in a ligand-dependent manner.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter. GABA receptor type A (GABAAR) possesses binding sites for a large group of pharmacological agents which are supposed to interact allosterically with each other. The aim of this work was to study the interaction between the positive allosteric modulators (PAMs) and the competitive antagonists of GABAARs. The GABA-induced chloride current (IGABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique. PAMs, neurosteroid allopregnanolone (Allo) and zolpidem (Zolp), a drug that positively modulates the GABAAR through interaction with the benzodiazepine (BDZ) site, doubled the IGABA amplitude in the control solution. Competitive antagonist of GABAARs, bicuculline (Bic, 5 µM) blocked the IGABA by 90%. The addition of 1 µM Allo or 0.5 µM Zolp to the Bic solution caused an unblocking effect, so that the IGABA amplitude increased 10 and 4 times from control value, correspondingly. This unblocking effect developed slowly, as evidenced by a threefold increase in the current rise time. Competitive antagonist of GABAARs, gabazine (GBZ, 0.5 µM) blocked the IGABA by 87%. The addition of 1 µM Allo to the GBZ solution caused an unblocking effect, so that the IGABA amplitude increased 7-fold. However, the addition of 0.5 µM Zolp to the GBZ solution did not cause an unblocking effect. So, Allo appeared to have a stronger unblocking potential than Zolp, and Bic binding site showed a higher sensitivity to the action of unblocking PAMs than GBZ binding site. The results indicate for the first time the existence of an allosteric relationship between the sites binding PAMs and the competitive antagonists of GABAAR.

Neurosteroids as Selective Inhibitors of Glycine Receptor Activity: Structure-Activity Relationship Study on Endogenous Androstanes and Androstenes.

The ability of androstane and androstene neurosteroids with modifications at C-17, C-5, and C-3 (compounds 1-9) to influence the functional activity of inhibitory glycine and γ-aminobutyric acid (GABA) receptors was estimated. The glycine- and GABA-induced chloride current (I Gly and I GABA) were measured in isolated pyramidal neurons of the rat hippocampus and isolated rat cerebellar Purkinje cells, correspondingly, using the patch-clamp technique. Our results demonstrate that all the nine neurosteroids display similar biological activity, namely, they strongly inhibited I Gly and weakly inhibited I GABA. The threshold concentration of neurosteroids inducing effects on I Gly was 0.1 µM, and for effects on I GABA was 10-50 µM. Moreover, our compounds accelerated desensitization of the I Gly with the IC50 values varying from 0.12 to 0.49 µM and decreased the peak amplitude with IC50 values varying from 16 to 22 µM. Interestingly, our study revealed that only compounds 4 (epiandrosterone) and 8 (dehydroepiandrosterone) were able to cause a significant change in I GABA in 10 µM concentration. Moreover, compounds 3 (testosterone), 5 (epitestosterone), 6 (dihydroandrostenedione), and 9 (etiocholanedione) did not modulate I GABA up to the concentration of 50 µM. Thus, we conclude that compounds 3, 5, 6, and 9 may be identified as selective modulators of I Gly. Our results offer new avenues of investigation in the field of drug-like selective modulators of I Gly.

The binding of donepezil with external mouth of K+-channels of molluscan neurons.

Earlier, we have shown a strong inhibitory effect of donepezil on K+-current of molluscan neurons (Solntseva et al., Comp Biochem Physiol 144, 319-326, 2007). In the present work, a possible interaction of donepezil with the external mouth of the channel was examined using, as a tool, tetraethylammonium (TEA), a classical antagonist of potassium channels. Experiments were conducted in isolated neurons of snail Helix aspersa using the two-microelectrode voltage-clamp technique. A high-threshold slow-inactivating K+-current involving Ca2+-dependent (I (C)) and Ca2+-independent (I (K)) components was recorded. The I (C) was estimated at 30 mV, and I (K) at 100 mV. The IC(50) values for blocking effect of donepezil on I (C) varied from 5.0 to 8.9 microM in different cells. Corresponding values for I (K) varied from 4.9 to 9.9 microM. The IC(50) values for blocking effect of TEA on I (C) lied in the range of 200 to 910 microM, and on I (K) lied in the range of 100 to 990 microM. The comparison of the effects of donepezil and TEA on the same cells revealed significant correlation between IC(50) values of these effects. The value of Spearman coefficient of correlation (r) was 0.77 for I (C) (P < 0.05), and 0.82 for I (K) (P < 0.05). In the presence of TEA, the effect of donepezil, both on I (C) and I (K), appears significantly weaker than in control solution. Dose-response curves of donepezil effect both on I (C) and I (K) were shifted right along horizontal axis when donepezil was applied in combination with TEA. Results suggest that TEA interferes with donepezil and precludes the occupation by donepezil of its own site. We suppose that the site for donepezil is situated near the TEA site with possible overlap.

Modulation of GABA and glycine receptors in rat pyramidal hippocampal neurones by 3α5ß-pregnanolone derivatives.

The ability of pregnanolone glutamate (PA-Glu), pregnanolone hemisuccinate (PA-hSuc) and pregnanolone hemipimelate (PA-hPim), neuroactive steroids with a negative modulatory effect on excitatory N-methyl-d-aspartate receptors, to influence the functional activity of inhibitory γ-aminobutyric acid and glycine receptors was estimated. The GABA- and glycine-induced chloride currents (IGABA and IGly) were measured in isolated pyramidal neurons of the rat hippocampus using the patch-clamp technique. Compound PA-Glu was found to potentiate IGABA and to inhibit IGly, while PA-hSuc and PA-hPim inhibited both IGABA and IGly. Moreover, PA-Glu, PA-hSuc, and PA-hPim had a greater effect on desensitization than on the peak amplitude of IGly. At a high concentration of glycine (500 µM), the effect of neurosteroids on the peak amplitude of IGly disappeared, and the acceleration of desensitization remained. The conversion of PA-Glu into androstane glutamate (AND-Glu), an analogue that lacks the C-17 acetyl moiety, completely eliminated the effects on these receptors. Our results indicate that the C-17 acetyl moiety is crucial for the action on IGABA and IGly. Our results indicate that the pregnanolone derivatives, in contrast to the androstane analogues, modulate IGABA and IGly at low micromolar concentrations and this family of neurosteroids can be useful for future structure-activity relationship studies of the steroid modulation of other receptor types.

The effects of ferric iron on voltage-gated potassium currents in molluscan neurons.

In isolated neurons of Helix pomatia, a two-microelectrode voltage clamp technique was used to study the effect of Fe3+ on voltage-gated potassium currents: a low-threshold fast-inactivating current (I(A)) and a high threshold slow-inactivating current with calcium-dependent (I(C)) and calcium-independent (I(DR)) components. Extracellular application of FeCl3 rapidly, reversibly and dose-dependently reduced the amplitude of I(A), I(C) and I(DR) with IC50 values of 49, 45 and 70 microM, respectively. Complete inhibition of K+ currents was reached at 100-500 microM Fe3+. The threshold for the total slow-inactivating potassium current shifted in a positive direction by 10-30 mV in the presence of Fe3+ (50-300 microM). Our work is the first demonstration of the strong blocking effect of Fe3+ on potassium currents of neuronal membrane.

Use-dependent inhibition of glycine-activated chloride current in rat neurons by ß-amyloid peptide pretreated with hexafluoroisopropanol.

Hexafluoroisopropanol (HFIP) is a nonpolar organic solvent that is often used to prepare ß-amyloid peptide (Aß) samples. In this work, we compare the effects of two different species derived from synthetic Aß1-42 and prepared without HFIP (Aß) or using HFIP (Aß/HFIP) on the glycine-activated chloride current (IGly). The experiments were conducted on the pyramidal neurons isolated from CA3 region of rat hippocampus. Transmembrane currents were recorded using a conventional patch-clamp technique in the whole-cell configuration. The IGly was induced by a step application of the agonist for 600 ms through glass capillary. Aß or Aß/HFIP was coapplied with glycine. The effects of the two species of the peptide have similar and distinctive features. Both substances caused a reduction in the peak amplitude and an acceleration of desensitization of the IGly. At the same time, the effect of Aß/HFIP was found to develop and recover more slowly and required several repeated applications for its saturation (use dependence). The effect of Aß/HFIP was voltage independent and equally pronounced at negative and positive membrane potentials. First, our results confirm that HFIP pretreatment may influence the properties of Aß. Second, new information on the glycine receptor ability to interact with drugs in use-dependent mode was obtained.

Glycine receptor in hippocampal neurons as a target for action of extracellular cyclic nucleotides.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are well known intracellular second messengers. At present study, we describe the effects of extracellularly applied cAMP and cGMP on glycine-induced chloride currents (I(Gly)) in isolated rat hippocampal pyramidal neurons. 50 or 500 µM glycine was applied for 600 ms with 1 min intervals. cAMP and cGMP were co-applied with glycine. We found that both cAMP and cGMP rapidly, reversibly and in a dose-dependent manner accelerated the I(Gly) desensitization. The effect was more prominent on I(Gly) induced by 500 µM than by 50 µM glycine. Dose-response curves were constructed in the 0.1-100,000 nM range of cAMP and cGMP concentrations. They demonstrate that threshold concentration of both compounds was about 1 nM and maximal effect was manifested at 100 nM. When cAMP and cGMP were added to the recording pipette, their extracellular application caused the effects similar to those obtained with normal intracellular medium. The effects of cyclic nucleotides remained unchanged in the presence of the antagonist of adenosine receptors in extracellular solution, and the agonist of adenosine receptors did not mimic the effect of cyclic nucleotides. The changes in the decay kinetics were equally pronounced at negative and positive membrane potentials. When co-administered 1 nM cAMP and 1 nM cGMP caused a weaker effect than either of the compounds alone which suggests a negative interaction between binding sites for cAMP and cGMP. This work describes a novel mode of action of cyclic nucleotides, namely, the modulation of GlyRs functions from extracellular side.

Donepezil is a strong antagonist of voltage-gated calcium and potassium channels in molluscan neurons.

Donepezil is an acetylcholinesterase inhibitor used in Alzheimer's disease therapy. The neuroprotective effect of donepezil has been demonstrated in a number of different models of neurodegeneration including beta-amyloid toxicity. Since the mechanisms of neurodegeneration involve the activation of both Ca(2+)- and K(+)-channels, the study of donepezil action on voltage-gated ionic currents looked advisable. In the present study, the action of donepezil on voltage-gated Ca(2+)- and K(+)-channels was investigated on isolated neurons of the edible snail (Helix pomatia) using the two-microelectrodes voltage-clamp technique. Donepezil rapidly and reversibly inhibited voltage activated Ca(2+)-current (I(Ca)) (IC(50)=7.9 microM) and three types of high threshold K(+)-current: Ca(2+)-dependent K(+)-current (I(C)) (IC(50)=6.4 microM), delayed rectifier K(+)-current (I(DR)) (IC(50)=8.0 microM) and fast transient K(+)-current (I(Adepol)) (IC(50)=9.1 microM). The drug caused a dual effect on low-threshold fast transient K(+)-current (I(A)), potentiating it at low (5 microM) concentration, but inhibiting at higher (7 microM and above) concentration. Donepezil also caused a significant hyperpolarizing shift of the voltage-current relationship of I(Ca) (but not of any type of K(+)-current). Results suggest the possible contribution of the blocking effect of donepezil on the voltage-gated Ca(2+)- and K(+)-channels to the neuroprotective effect of the drug.

Cyclic nucleotides induce long-term augmentation of glutamate-activated chloride current in molluscan neurons.

1. Literature data indicate that serotonin induces the long-term potentiation of glutamate (Glu) response in molluscan neurons. The aim of present work was to elucidate whether cyclic nucleotides can cause the same effect. 2. Experiments were carried out on isolated neurons of the edible snail (Helix pomatia) using a two-microelectrode voltage-clamp method. 3. In the majority of the cells examined, the application of Glu elicited a Cl- -current. The reversal potential (Er) of this current lied between -35 and -55 mV in different cells. 4. Picrotoxin, a blocker of Cl- -channels, suppressed this current equally on both sides of Er. Furosemide, an antagonist of both Cl- -channels and the Na+/K+/Cl- -cotransporter, had a dual effect on Glu-response: decrease in conductance, and shift of Er to negative potentials. 5. A short-term (2 min) cell treatment with 8-Br-cAMP or 8-Br-cGMP caused long-term (up to 30 min) change in Glu-response. At a holding potential of -60 mV, which was close to the resting level, an increase in Glu-activated inward current was observed. This potentiation seems to be related to the right shift of Er of Glu-activated Cl- -current rather than to the increase in conductance of Cl- -channels. The blocking effect of picrotoxin rested after 8-Br-cAMP treatment. 6. The change in the Cl- -homeostasis as a possible mechanism for the observed effect of cyclic nucleotides is discussed.

Selective suppression of the slow-inactivating potassium currents by nootropics in molluscan neurons.

The role of the voltage-gated K+ channels in the effect of some nootropics was investigated. Earlier, the multiple effect of high concentrations of two nootropics, piracetam and its peptide analogue GVS-111 [Seredenin et al. (1995), US Patent No. 5,439,930], on Ca2+ and K+ currents of molluscan neurons was shown [Solntseva et al. (1997), General Pharmacology 29, 85-89]. In the present work, we describe the selective effect of low concentrations of these nootropics as well as vinpocetine on certain types of K+ current. The experiments were performed on isolated neurons of the land snail Helix pomatia using a two-microelectrode voltage-clamp method. The inward voltage-gated Ca2+ current (ICa) and three subtypes of the outward voltage-gated K+ current were recorded: Ca2+-dependent K+ current (IK(Ca)), delayed rectifying current (IKD), and fast-inactivating K+ current (IA). It has been found that I Ca was not changed in the presence of 30 microM vinpocetine, 100 microM piracetam or 10 nM GVS-111, while slow-inactivating, TEA-sensitive IK(Ca) and IKD were inhibited (IK(Ca) more strongly than IKD). In contrast, the fast-inactivating, 4-AP-sensitive K+ current (IA) was not diminished by low concentrations of piracetam and GVS-111, while vinpocetine even augmented it. A possible role of slow-inactivating subtypes of the K+ channels in the development of different forms of dementia is discussed.