Allosteric modulation of ligand gated ion channels by ivermectin.

Ivermectin acts as a positive allosteric regulator of several ligand-gated channels including the glutamate-gated chloride channel (GluCl), gamma aminobutyric acid type-A receptor, glycine receptor, neuronal alpha7-nicotinic receptor and purinergic P2X4 receptor. In most of the ivermectin-sensitive channels, the effects of ivermectin include the potentiation of agonist-induced currents at low concentrations and channel opening at higher concentrations. Based on mutagenesis, electrophysiological recordings and functional analysis of chimeras between ivermectin-sensitive and ivermectin-insensitive receptors, it has been concluded that ivermectin acts by insertion between transmembrane helices. The three-dimensional structure of C. elegans GluCl complexed with ivermectin has revealed the details of the ivermectin-binding site, however, no generic motif of amino acids could accurately predict ivermectin binding site for other ligand gated channels. Here, we will review what is currently known about ivermectin binding and modulation of Cys-loop receptor family of ligand-gated ion channels and what are the critical structural determinants underlying potentiation of the P2X4 receptor channel.

[Purinergic P2X family and specific features of the P2X7 subtype]. / Purinergní P2X rodina a specifické vlastnosti P2X7 podtypu.

Purinergic P2X receptors (P2XR), activated by extracellular adenosine 5'-triphosphate (ATP), represent a specific type of ligand-gated ion channels. They form functional trimeric homomers or heteromers which are nonselectively cation-permeable after receptor activation. P2X receptors are widely expressed in excitable and nonexcitable tissues and are involved in many physiological and pathophysiological processes such as platelet aggregation, contraction of smooth muscle, immune responses, cell proliferation and apoptosis or neurotransmission. In mammals, seven P2X subunits (P2X1-P2X7) have been identified. They differ mainly in distribution, pharmacological profile and kinetics of ATP-induced responses. The subtype P2X7 is the most specific in the P2X family and widely differs from other P2X subtypes.

Dependence of spontaneous electrical activity and basal prolactin release on nonselective cation channels in pituitary lactotrophs.

All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH(3) cells, replacement of extracellular Na(+) with large organic cations, but not blockade of voltage-gated Na(+) influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na(+) with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH(3) cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH(3) cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca(2+) influx and prolactin release in a concentration-dependent manner. Gd(3+), a common Ca(2+) channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca(2+) influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca(2+) influx and hormone release in lactotrophs and GH(3) cells.

Facilitation of glutamate and GABA release by P2X receptor activation in supraoptic neurons from freshly isolated rat brain slices.

The supraoptic nuclei (SON), the hypothalamic release site of vasopressin and oxytocin, receive a non-glutamatergic, excitatory input from the caudal medulla that uses noradrenaline and ATP as neurotransmitters. Here, we studied the actions of extracellular ATP on SON neurons in hypothalamic slices isolated from the brains of 16- to 24-day-old rats. Whole-cell current clamp recordings performed 1-6 h after isolation showed that exogenous ATP application increased the frequency of action potentials and induced the depolarization of resting membranes. Voltage clamp recordings showed that ATP increased the frequency of GABAergic or glutamatergic spontaneous synaptic currents without changing their amplitude and evoked inward current (126±13 pA) in about 80% of SON neurons. The application of ATPγS and 2MeSATP mimicked the effects of ATP, but 2MeSADP, 2MeSAMP and αßmeATP had no effect. The P2X7 receptor agonist, BzATP, did not induce an inward current, but it increased intracellular calcium concentration in non-neuronal SON cells in slices. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) inhibited ATP-induced currents, whereas pH 6.5 and ivermectin, a specific allosteric modulator of the P2X4 receptor, potentiated ATP-induced currents. The P2Y1-selective antagonist, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), had no effect on ATP-induced responses. Quantitative real-time PCR showed that P2X2>P2X7>P2X4 purinergic receptor mRNAs were expressed in the SON tissue, but the levels of P2X1, P2X3, P2X5, P2X6, P2Y1, P2Y2 and P2Y12 mRNA were minor. These results show that SON neurons express functional presynaptic and extrasynaptic P2X2 and P2X4 receptors that modulate glutamate and GABA release and control the electrical excitability of SON neurons.

Roles of conserved ectodomain cysteines of the rat P2X4 purinoreceptor in agonist binding and channel gating.

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.

GnRH-I and GnRH-II-induced calcium signaling and hormone secretion in neonatal rat gonadotrophs.

Two forms of gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are commonly present in mammals. The main hormone controlling reproduction is GnRH-I acting through its receptor (GnRHR-I), whereas the function of GnRH-II is unknown. In primates, it has been suggested that GnRH-II is a specific agonist for the structurally distinct GnRHR-II. Here we compared effects of GnRH-I and GnRH-II on intracellular calcium and gonadotropin hormone release in neonatal rat gonadotrophs in vitro and the dependence of agonist actions on cyclic nucleotide levels. Both agonists elevated intracellular calcium and stimulated gonadotropin secretion in a concentration-dependent manner, with comparable peak amplitudes, but GnRH-I was three times more potent than GnRH-II. Antide, a specific GnRHR-I antagonist, completely blocked the action of both agonists on gonadotropin release. Inhibition of adenylyl cyclase activity by melatonin and MDL significantly attenuated GnRH-I- and GnRH-II-induced calcium signaling and gonadotropin release, whereas inhibition of soluble guanylyl cyclase activity was ineffective. GnRH-II also generated calcium oscillations in a fraction of gonadotrophs not expressing melatonin receptors. These results indicate that GnRH-I and GnRH-II act on the same GnRHR to stimulate gonadotropin release through intracellular calcium and cyclic nucleotide signaling, and that GnRH-II is less potent agonist for this receptor in neonatal rat gonadotrophs.

Molecular structure of purinergic P2X receptors and their expression in the hypothalamus and pituitary.

Purinergic P2X receptors represent a novel structural type of ligand-gated ion channels activated by extracellular ATP. So far, seven P2X receptor subunits have been found in excitable as well as non-excitable tissues. Little is known about their structure, mechanism of channel opening, localization, and role in the central nervous system. The aim of this work is to summarize recent investigations and describe our contribution to elucidating the structure of the ATP binding site and transmembrane domains of the P2X receptor, we also discuss the expression and physiological roles played by the ATP and P2X receptors in the anterior pituitary and hypothalamus.

Characterization of neuromuscular transmission in mice with progressive motoneuronopathy.

Progressive motoneuronopathy (PMN) is an autosomal recessive mouse disease, which is characterized by the development of hind limbs paralysis rapidly progressing to the anterior parts of the body, muscular atrophy, respiratory depression, and death at 6-7 postnatal weeks. Here, we recorded the resting membrane potential (RMP), spontaneous miniature endplate potentials (MEPPs), and quantum content of endplate potentials (EPP) at the diaphragm muscle fibers in controls and PMN mice aged 18 to 43 days. In control animals, there was a progressive increase in RMP, MEPP frequency and EPP quantum content, as well as a decrease in mean MEPP amplitude. In PMN mice, the developmental increase in frequency and decrease in the amplitude of MEPPs was practically stopped at the postnatal day 18, whereas RMP increased but only until the age of 31 days and then progressively decreased. The distribution histogram of RMP in PMN mice older than 35 days revealed the existence of two subpopulations of muscle fibers: one showing a denervation-like decrease in RMP and the second, which was matching controls. In addition, EPP quantum content was significantly attenuated in older PMN animals. These results indicate that neurotransmission is severely affected in advanced, but not in early stage of disease, which is apparently due to a partial denervation of the muscles.

Melatonin action in neonatal gonadotrophs.

Neonatal pituitary cells express MT1 and MT2 subtype of melatonin receptors that are coupled to pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings have revealed that a reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores. Inhibition of calcium influx, probably in a cAMP/protein kinase C-dependent manner, and the accompanying calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. Melatonin-induced attenuation of GnRH-induced and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular pools attenuates the amplitude of calcium signal. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. During the development, such tonic inhibitory effects of melatonin on GnRH action gradually decline due to a decrease in expression of functional melatonin receptors. In adult animals, melatonin does not have obvious direct effects on pituitary functions, whereas the connections between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.

Dual effect of melatonin on gonadotropin-releasing-hormone-induced Ca(2+) signaling in neonatal rat gonadotropes.

In neonatal rat gonadotropes, melatonin inhibits gonadotropin-releasing-hormone (GnRH)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)); in cells transfected with the Mel1a melatonin receptor, however, melatonin has been shown to potentiate agonist-stimulated [Ca(2+)](i) increase. To elucidate this discrepancy, we investigated the effects of melatonin in neonatal gonadotropes over a wide range of melatonin concentrations. Nystatin perforated patch recording of Ca(2+)-dependent potassium currents was used to monitor GnRH-induced [Ca(2+)](i) changes. In 32% of cells, increasing melatonin concentrations in the range of 1 pM to 100 nM prolonged the latency of, and inhibited GnRH (10 nM)-stimulated [Ca(2+)](i) increases in a concentration-dependent manner. In the remaining 68% of cells, the Ca(2+) increase elicited by exposure to 10 nM GnRH was also inhibited by picomolar concentrations of melatonin, but at nanomolar concentrations the inhibitory effect disappeared and melatonin was only able to prolong the latency of the response. This dual effect of melatonin however was not observed in cells stimulated with lower (2 nM) GnRH concentrations; in that case, melatonin was inhibitory at all concentrations tested with an IC(50) of about 30 pM. In contrast, application of nanomolar concentrations of melatonin resulted in potentiation of the GnRH-induced Ca(2+) increase in a small population of gonadotropes which did not respond by inhibition or prolonged latency. These results indicate that in neonatal gonadotropes, melatonin has both inhibitory and potentiating effects on GnRH-stimulated [Ca(2+)](i) increases. Ranges of concentrations needed to produce either effect suggest that two distinct G proteins may be involved, as already observed in transfected cells.

Differences in gonadotropin-releasing hormone-induced calcium signaling between melatonin-sensitive and melatonin-insensitive neonatal rat gonadotrophs.

The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that there are two pathways for initiation of GnRH-induced calcium signaling in neonatal gonadotrophs. The first pathway is mediated by inositol 1,4,5,-trisphosphate production, whereas the second involves extracellular calcium entry through voltage-dependent L-type Ca2+ channels, protein kinase C activation, and Ca2+ release from a ryanodine-sensitive store, which may coactivate Ca2+ release from an inositol 1,4,5,-trisphosphate-sensitive store. Only the second mechanism is accessible to inhibition by melatonin.

Melatonin inhibits GnRH-induced Ca2+ mobilization and influx through voltage-regulated channels.

In neonatal rat gonadotrophs, melatonin acts through the high-affinity membrane-bound receptors to inhibit GnRH-induced [Ca2+]i increase. GnRH increases [Ca2+]i primarily by mobilization from the inositol trisphosphate-sensitive pool followed by Ca2+ influx through the voltage-sensitive channels. Melatonin inhibits the GnRH-induced [Ca2+]i increase. When added after the GnRH-induced spike, melatonin decreases [Ca2+]i in 52% of the gonadotrophs. The effect of melatonin is dependent on extracellular Ca2+ and may be mimicked by Ca2+-free medium or verapamil. When added before GnRH, melatonin inhibits the [Ca2+]i spike. This effect of melatonin is independent of extracellular Ca2+ as it persists in Ca2+-free medium. These findings indicate that melatonin blocks Ca2+ mobilization as well as Ca2+ influx in the gonadotrophs.

Inhibitory effect of melatonin on gonadotropin-releasing hormone-induced Ca2+ oscillations in pituitary cells of newborn rats.

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from the anterior pituitary gland of 6- to 8-day-old rats. GnRH-induced [Ca2+]i oscillations were recorded indirectly by monitoring the activity of apamin-sensitive Ca(2+)-activated K+ channels using the perforated patch-clamp technique and fast microperfusion system. Melatonin (1 nM) inhibited the initiation or attenuated the amplitude of oscillatory current responses induced by 10 nM GnRH in 72% of GnRH-sensitive cells. Analysis of the melatonin dose-inhibition relationship showed that melatonin inhibited the initiation of [Ca2+]i oscillations with IC50 = 0.35 nM. In partially inhibited cells, melatonin reduced the GnRH-induced current amplitude by 55% on the average, prolonged the delay in onset of response to GnRH and decreased the frequency of oscillations. Once initiated by GnRH, the amplitude and frequency of oscillatory currents was inhibited by melatonin after a latency of 10-30 s. These effects of melatonin were fully reversible. After pretreatment of neonatal gonadotropes with pertussis toxin, no inhibition by melatonin was observed. The inhibitory effect of melatonin on initiation, amplitude and frequency of GnRH-induced oscillatory current persisted in the absence of external Ca2+. Melatonin alone did not induce any transmembrane current or membrane potential changes. These observations suggest that melatonin reduces GnRH-induced calcium mobilization from intracellular stores.

Copper modulation of NMDA responses in mouse and rat cultured hippocampal neurons.

The effect of Cu2+ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu2+ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu2+ directly inhibits NMDA receptors with an IC50 of 0.27 microM. Cu2+ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu2+ and glycine-Cu2+ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu2+ (10-100 microM) of responses to 10 microM NMDA was essentially voltage-independent. The onset of inhibition by 100 microM Cu2+ of responses to 2 microM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that Cu2+ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu2+ to NMDA receptors, calculated from the rate of onset of block by Cu2+ of test responses to NMDA, was 19 times slower than in the presence of 30 microM NMDA, suggesting that Cu2+ interacts preferentially with agonist-bound receptors. Our results show that Cu2+ is a potent inhibitor of NMDA receptor-mediated responses.

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 effects of calcium and calcium channel blockers on sodium pump.

The effects of 10 mM Ca2+ and Ca2+ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na+, K+ pump activity of mouse diaphragm muscle fibres enriched with Na+ were compared with the changes in cytosolic [Ca2+]. The electrogenic Na+ pump activity produced by adding K+ to muscles previously bathed for 4 h in a K(+)-free, 2-mM [Ca2+] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca2+. Verapamil 10(-5) M, 10(-5) M diltiazem and 10(-7) M flunarizine effectively prevented the effect of elevated [Ca2+]. At these concentrations, these drugs did not affect the K(+)-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca2+] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6- aminobenzofuran-5-oxy]2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N '- tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261 +/- 6 nM. After 4 h in a Liley K(+)-free, 2 mM [Ca2+] solution, the cytosolic [Ca2+] increased to 314 +/- 28 nM. Increase in [Ca2+] from 2 to 10 mM caused a twofold increase of cytosolic [Ca2+] to 637 +/- 26 nM. This rise was, like the Ca(2+)-induced inhibition of electrogenic pump, prevented by 10(-5) M verapamil, 10(-5) M diltiazem and 10(-7) M flunarizine. The results suggest that substances which block Ca2+ entry into the cell prevent the Ca(2+)-induced inhibition of the Na+ pump.

7-Oxo-prostacyclin affects the electrogenic Na+/K+ pump in mouse diaphragm fibers.

The effect of a stable prostacyclin derivative, 7-oxo-prostacyclin, on the electrogenic Na+/K+ pump in mouse diaphragm muscle was investigated. Resting membrane potentials of muscle cells were measured with glass microelectrodes. In fresh diaphragm preparations from 7-oxo-prostacyclin-pretreated mice (50 micrograms.kg-1 7-oxo-prostacyclin i.p. 30-34 h prior to the experiment) resting membrane potentials were significantly higher (-83.86 +/- 1.40 mV, mean +/- S.E.M.) than in control preparations (-70.78 +/- 1.07 mV). Ouabain at a concentration of 10(-4) mol.l-1 abolished this hyperpolarization (P < 0.05). The electrogenic effect induced by addition of 5 mmol.l-1 K+ to Na(+)-loaded muscles from 7-oxo-prostacyclin-pretreated animals was also enhanced (delta resting membrane potential = 21.73 +/- 3.13 mV) compared with that of the controls (delta resting membrane potential = 18.36 +/- 1.29 mV). Ouabain antagonized the increase in electrogenicity of Na(+)-loaded diaphragms induced by 7-oxo-prostacyclin pretreatment (P < 0.05). In contrast to control preparations, no inhibition of the electrogenic effect induced by 10 mmol.l-1 Ca2+ ions in Na(+)-loaded muscles was observed in diaphragms from 7-oxo-prostacyclin-pretreated animals. In acute experiments with Na(+)-loaded muscles, where 10(-7) mol.l-1 7-oxo-prostacyclin was added to the bath, resting membrane potential reached up to -100 mV. The electrogenic pump-induced increase in resting membrane potential amounted to approximately 30 mV. This effect could be also abolished by 10(-4) mol.l-1 ouabain (P < 0.05).(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.

Potentiation of GABAA receptor in cultured mouse hippocampal cells by brain-derived peptide mixture cerebrolysin.

Application of Cerebrolysin (0.1 microgram per 1 ml) by a fast microperfusion system induced an inward current of 0.2 to 1 nA in all neurones from newborn mouse hippocampi held at -30 mV membrane potential. Cerebrolysin-induced currents were reduced by the GABAA antagonist bicuculline (2 microM) by 65%, by the NMDA antagonist aminophosphovaleric acid (APV, 10 microM) by 27% and by the non-NMDA antagonist cyanonitriquinoxalinedione (CNQX, 10 microM) by 20%. Cerebrolysin dialyzed through a 3.6 kD gut did not induce any transmembrane current but potentiated the response induced by GABA (10 microM) to 135%. We conclude that, in addition to amino acids which activate GABAA, NMDA and non-NMDA receptors, Cerebrolysin also contains a peptide which potentiates the GABAA receptor response.

Effect of ivermectin on gamma-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones.

The effect of ivermectin on gamma-aminobutyric acid (GABA)-induced Cl- currents was studied in embryonicse hippocampal cells in culture. When 0.1 microM ivermectin was applied to the perfusion medium, the responses to 2 microM GABA were enhanced to 273% within 60 s, and the GABA EC50 was reduced from 8.2 to 3.2 microM. Half-maximal potentiation of GABA responses was found with 17.8 nM ivermectin. The potentiating effect of ivermectin diminished to 146% within 10 min but the GABA EC50 did not change any further. At the same time, the maximal GABA-induced Cl- current decreased to 64%. Both the fast and slow desensitization time constants of GABA-activated membrane currents were shortened after ivermectin application. The final effect of ivermectin was irreversible. Modulation of the GABA responses by ivermectin did not interfere with the potentiation induced by diazepam and pentobarbital or with the sensitivity to blockade by bicuculline, picrotoxin and Zn2+. These results support the view that ivermectin binds to a novel site on the GABAA receptor and allosterically enhances the affinity of the GABA binding site. The more slowly occurring conformational changes in the ivermectin-GABAA receptor complex apparently accelerate the desensitization of the GABAA receptor, reducing the amplitude of maximal GABA-induced currents.