Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 microM) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 microM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10-100 nM), an activator of protein kinase C (PKC), but not 4-alpha-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 microM), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.     

Expression of functional nicotinic acetylcholine receptors in neuroepithelial bodies of neonatal hamster lung

Pulmonary neuroepithelial bodies (NEB) are presumed airway chemoreceptors involved in respiratory control, especially in the neonate. Nicotine is known to affect both lung development and control of breathing. We report expression of functional nicotinic acetylcholine receptors (nAChR) in NEB cells of neonatal hamster lung using a combination of morphological and electrophysiological techniques. Nonisotopic in situ hybridization method was used to localize mRNA for the beta 2-subunit of nAChR in NEB cells. Double-label immunofluorescence confirmed expression of alpha 4-, alpha 7-, and beta 2-subunits of nAChR in NEB cells. The electrophysiological characteristics of nAChR in NEB cells were studied using the whole cell patch-clamp technique on fresh lung slices. Application of nicotine ( approximately 0.1-100 microM) evoked inward currents that were concentration dependent (EC50 = 3.8 microM; Hill coefficient = 1.1). ACh (100 microM) and nicotine (50 microM) produced two types of currents. In most NEB cells, nicotine-induced currents had a single desensitizing component that was blocked by mecamylamine (50 microM) and dihydro-beta-erythroidine (50 microM). In some NEB cells, nicotine-induced current had two components, with fast- and slow-desensitizing kinetics. The fast component was selectively blocked by methyllcaconitine (MLA, 10 nM), whereas both components were inhibited by mecamylamine. Choline (0.5 mM) also induced an inward current that was abolished by 10 nM MLA. These studies suggest that NEB cells in neonatal hamster lung express functional heteromeric alpha 3 beta 2, alpha 4 beta 2, and alpha 7 nAChR and that cholinergic mechanisms could modulate NEB chemoreceptor function under normal and pathological conditions.     

Action of cyclic nucleotide phosphodiesterase inhibitors on the acetylcholine potential

A study was made of the action of theophylline, isobutyrylmethylxanthine and caffeine on the sensitivity of mouse diaphragmatic muscle fibers to iontophoretically applied acetylcholine (ACh). It was shown that these substances at concentrations of 5 X 10(-4) -5 X 10(-3) M reduced the amplitude and increased the duration of the ACh potential as well as accelerated desensitization of the cholinoceptor at repetitive application of ACh. As regards the action on the ACh potential amplitude two phases which differed in the time-course of development and washing were recognized: rapid and slow. Addition of dibutyryl-cAMP (5 X 10(-4) M) after theophylline (10(-3) M) potentiated the latter's action on the ACh potential amplitude but did not influence its duration and the rate of desensitization. It is assumed that the action of phosphodiesterase inhibitors on the duration of the ACh potential and the rate of desensitization is not mediated by an elevation in the muscle cAMP content. Apparently, cAMP accumulation may be responsible but for the phase of a slow decrease in the ACh potential amplitude.     

Direct measurement of the concentration- and time-dependent open probability of the nicotinic acetylcholine receptor channel.

Using the outside-out patch clamp recording technique together with a rapid solution exchange system, we measured ionic currents through nicotinic acetylcholine (ACh) receptor channels from BC3H-1 cells in response to rapid applications of 0.3-1,000 microM ACh. We used nonstationary fluctuation analysis of ensembles of responses to deduce the number of channels in the patch, the maximum open channel probability as a function of ACh concentration and the time course of a fast desensitization process. We found that: (a) Excised patches from BC3H-1 cells typically contain between 50 and 150 functional ACh receptor ion channels. (b) The open channel probability is proportional to [ACh]1.95 at low concentrations of ACh, is half-maximal at 20 microM ACh and saturates above 100 microM ACh. (c) ACh is a very efficacious agonist; 100 microM ACh opens at least 90% of the available channels. This estimate of efficacy is model-independent. (d) The rate of decay of the agonist-induced current is concentration-dependent. In the presence of 100 microM ACh the current decays with a time constant of 50-100 ms. It decays more slowly in the presence of lower concentrations of agonist but is relatively insensitive to voltage.     

Effects of Aliphatic Alcohols and Food Additives on Nicotinic Acetylcholine Receptors in Xenopus Oocytes

To study the effects of food additives on nicotinic acetylcholine receptors (nAChR), they were expressed in Xenopus oocytes that received an injection of mRNA prepared from electroplax of Electrophorus electricus. The response of nAChR elicited by acetylcholine (ACh) was measured electrophysiologically in the presence and absence of aliphatic alcohols and food additives. All compounds examined inhibited nAChR non-competitively in a concentration-dependent way. The inhibition was stronger when the inhibitors were perfused lmin before ACh, than when they were perfused simultaneously with ACh. The inhibition of nAChR by aliphatic alcohols (propanol to hexanol) increased as the number of carbon chains increased. The addition of alcohols and food additives did not affect the desensitization of nAChR caused by 2 μm ACh. These results suggest that alcohols and food additives bind to the anesthetic binding site in nAChR and inhibit it noncompetitively. However, these compounds will not hinder signal transmission in neuromuscular junctions under physiological conditions, because their inhibition constants are more than 1 mm and muscles usually have more receptors than the number necessary for signal transmission.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O- tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.     

Acetylcholine receptor channel properties in rat myotubes exposed to forskolin

Rat myotubes exposed to forskolin were studied by patch-clamp technique in cell-attached single channel recording configuration. Channel open time and opening frequency of the main class of acetylcholine receptor- (AChR-) channels (accounting for more than 90% of all unitary events) decreased in the presence of forskolin (20-100 microM). The forskolin-induced action on the AChR function fully developed with a delay of 30-60 minutes from the peak of cytosolic cyclic AMP (cAMPi) concentration. Both cAMP (1 mM), applied intracellularly for 10 min, and dibutyryl cAMP (0.5 mM), applied extracellularly for 90 min, did not accelerate the rate of desensitization of myotubes studied in whole-cell patch-clamp recording configuration. It was concluded that the action of forskolin on AChR-channel function of rat myotubes could be not associated with the cAMPi-dependent phosphorylation of AChR.     

Effects of concanavalin A on desensitization kinetics of GABA responses inAchatina fulica neurons

The effects of the lectin concanavalin A (Con A), on the kinetics of desensitization of the responses of voltage clampedAchatina fulica LP5 neuron to microperfused acetylcholine (ACh) and GABA were compared. Both ACh and GABA elicited increases in chloride conductance which decayed biphasically during prolonged applications of these agonists; an initial rapid decay was followed by a later slow decay. Con A (5 g/ml) accelerated both the fast and the slow decays of responses to ACh. Con A (5 g/ml) also accelerated the fast decay of responses to GABA, but the slow decay was unaffected, even by 20 g/ml or more of the lectin. It is suggested that, at least in the case of GABA receptor, the fast and slow decays involve distinct desensitization kinetics. The effects of Con A on the desensitization of the ACh and GABA responses were reversed byd-mannose, a competitive and specific inhibitor of Con A binding to membrane sugar residues. These results provide further evidence that receptor desensitization can be influenced by perturbing the sugar moieties associated with the subunits comprising these signalling macromolecules. The carbohydrate residues may play an important role in regulating desensitization of transmitter receptors.Abbreviations ACh acetylcholine - Con A concanavalin A     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.     

Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in alpha(1)-adrenergic pathway via the modulation of acetylcholine-activated K(+) channels in mouse atrial myocytes

We have investigated the effect of alpha(1)-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K(+) currents (I(KACh)). I(KACh) was recorded in mouse atrial myocytes using the patch clamp technique. I(KACh) was activated by 10 microm ACh and the current decreased by 44.27 +/- 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 +/- 2.22% (n = 9), indicating the presence of PE-induced desensitization. I(KACh) was fully recovered from desensitization after a 6-min washout. PE-induced desensitization of I(KACh) was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP(2)) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization of I(KACh) in the presence of PE was further increased (97.25 +/- 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of I(KACh) at the second exposure after washout was reduced to 19.65 +/- 2.61% (n = 6) of the preceding level. These data suggest that the K(ACh) channel is modulated by PE through PLC stimulation and depletion of PIP(2).     

Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors

In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]_i). Whole‐cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]_i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP‐induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 487–501, 2013     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

Desensitization of nicotinic acetylcholine receptors in central nervous system neurons of the stick insect (Carausius morosus) by imidacloprid and sulfoximine insecticides

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 10⁻⁴ M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, I_ACh1 and I_ACh2 respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, I_ACh1H and I_ACh2H with IC₅₀s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, I_ACh2L, with an IC₅₀ of 2.6 nM, while a component of the fast-desensitizing current, I_ACh1L, was least sensitive, with an IC₅₀ of 81 nM I_ACh1L appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized I_ACh1H and both slowly desensitizing components, with IC₅₀s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects.     

Glutamate receptor desensitization and its role in synaptic transmission

Responses of excitatory amino acid receptors to rapidly applied glutamate were measured in outside-out membrane patches from chick spinal neurons. The peak current varied with glutamate concentration, with a half-maximal response at 510 microM and a Hill coefficient near 2. Currents activated by 1 mM glutamate desensitized and recovered in two phases. The faster time constant was identical to the time constant of decay of synaptic currents, suggesting that glutamatergic synaptic currents are terminated, in part, by receptor desensitization. Steady-state desensitization was evident following application of only 2-3 microM glutamate, concentrations comparable to levels in the extracellular space in the intact brain. Thus, glutamate receptor desensitization can affect synaptic efficacy in two ways: at high concentrations, rapid desensitization of receptors may curtail synaptic currents; at low concentrations, there is a significant reduction in the number of activatable receptors.     

Effects of "non-quantal" acetylcholine on miniature endplate currents in mice

The possibility of postsynaptic potentiation (PSP) and desensitization developing due to nonquantal acetylcholine (ACh) secretion was investigated in mouse diaphragm with reference to time-amplitude relationships of miniature endplate currents (MEPC). The H effect (which characterizes nonquantal secretion (NS) of ACh) fell to zero over 3 h under the action of armine-induced inhibition of acetylcholinesterase (AChE) at a temperature of 20°C. A decline in the decay time constant () of MEPC unaccompanied by observable alteration in MEPC amplitude occurred at the same time. This accelerated decay of MEPC was not observed in the absence of NS (the early stages of denervation). Start of NS did not show any effect on maximum retardation of MEPC decay due to AChE inhibition, indicating that no PSP sets in under the effects of non-quantal secretion. The effect of decline in accelerated with a rise in temperature; it could be reproduced with neostigmine replacing armine, while remained unchanged in the time spells investigated with AChE in its active state. Non-quantal ACh is not thought to produce substantial retardation of MEPC decay, although it does bring about desensitization, signs of which may be partially masked owing to concurrent onset of PSP.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 507–513, July–August, 1990.     

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Possibility of autocrine beta-adrenergic signaling in C2C12 myotubes

Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by insulin (P < 0.05) and increased cAMP concentrations (P < 0.05). The decrease in insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a beta-adrenergic antagonist) and prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine beta-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism.     

Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers

(S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2).     

A model for the secondary structure of beta-lactamases

The effects of the quaternary agent meproadifen on ACh-activated channel currents were studied on myoballs cultured from hind limb muscles of neonatal rats. Meproadifen (0.02-0.1 microM) combined with ACh (0.1-0.3 microM) in the patch pipette caused an increase, followed by a decrease, in the frequency of channel openings. At concentrations greater than 0.2 microM the initial phase was not detected and a rapid and marked reduction in the opening frequency was observed. Meproadifen (up to 2.5 microM) produced no change in the duration or conductance of the open state of ACh-activated channels. In addition, this agent induced the appearance of events with a marked increase in the 'noise' during the opening phase. The lack of effect under inside-out patch conditions suggested that meproadifen binds to a site located at the external portion of the nicotinic macromolecule and has no access to it through the cell membrane. This study indicated that non-competitive antagonists such as meproadifen can facilitate receptor activation and desensitization.