Block by acetylcholine of mouse muscle nicotinic receptors,stably expressed in fibroblasts

We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence. This is in contrast to the strong voltage dependence observed for the degree of block. Deviations from the predictions of the simple model are seen in data collected at positive transmembrane potentials and at negative potentials for ACh concentrations < 1 mM. Less concentration dependence is observed than expected. Of a number of models tested, we demonstrate that two models incorporating both a high and a low affinity blocking site can predict our data.     

Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations

We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.     

Photoresponsive nanogels formed by the self-assembly of spiropyrane-bearing pullulan that act as artificial molecular chaperones

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation.     

Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors

Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.     

Untangling ligand induced activation and desensitization of G-protein-coupled receptors

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.     

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.     

The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor.

When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing.     

Activation and desensitization of heteromeric neuronal nicotinic receptors: implications for non-synaptic transmission

Consideration of the activation and desensitization properties of neuronal nicotinic acetylcholine receptors (nAChRs) predicts that there should be a range of concentrations over which low ambient levels of agonist can continuously open nAChR channels. These findings support the idea that postsynaptic nAChRs may participate in unconventional cellular signaling mediated by the release of acetylcholine from diffusely distributed non-synaptic cholinergic varicosities.     

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.     

The role of nicotinic receptors in B-lymphocyte development and activation

We studied the binding of [(3)H]-epibatidine and [(125)I-]alpha-bungarotoxin, as well as subunit-specific antibodies with purified B lymphocytes of C57Bl/6J mice and found that these cells contained 12,200+/-3200 of alpha4(alpha5)beta2 and 3130+/-750 of alpha7(alpha5beta4) nicotinic acetylcholine receptors per cell. According to flow cytometry data, the highest expression of alpha4(alpha5)beta2 receptors was observed in immature newly generated B lymphocytes of the bone marrow, while the number of alpha7(alpha5beta4) receptors grew up along with the B cell maturation in the spleen. By using alpha4, beta2 or alpha7 knockout and chimera mice, it was shown that both receptor subtypes supported the survival of B cell precursors and increased the size of B-lymphocyte population in the bone marrow. In contrast, propagation of mature B lymphocytes in the spleen was controlled by alpha7-containing subtype only. Moreover, mature B lymphocytes became sensitive to nicotine only in the absence of beta2-containing receptors. Knockout mice had less serum IgG, IgG-producing cells and natural IgG antibodies than their wild-type counterparts, while the absence of beta2-containing receptors resulted in increased B-lymphocyte activation and antibody immune response. The data obtained indicate that nicotinic receptors are involved in regulating B-lymphocyte development and activation, possibly, by affecting expression and/or signaling of CD40, the two subtypes playing different roles.     

Concomitant protein phosphorylation and endogenous acetylcholine release induced by nicotine: Dependency on neuronal nicotinic receptors and desensitization

Summary 1. Nicotine stimulated two Ca²⁺-dependent processes in rat frontal cortex synaptosomes: the phosphorylation of an 80-kDa protein band and the release of endogenous ACh.³ Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptosomal plasma membrane induced by the drug. Changes in the state of phosphorylation of the 80-kDa band (presumed to contain synapsin I) were correlated with changes in the release of ACh as follows, from 2 to 4.2. Blockade of predominant, nerve terminal P-type Ca²⁺ channels with -agatoxin-IVA, did not prevent nicotine from stimulating ACh release. In contrast, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylation induced by either nicotine or veratridine. Taken together, these data suggest that, upon nicotine stimulation, Ca²⁺ enters nerve terminals through two distinct pathways. The first, via Ca²⁺ channels, is necessary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotine-induced phosphorylation and release, is the neuronal nAChR itself.3. Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosphorylation. This shows that diminished release is associated to decreased phosphorylation of the 80-kDa protein band, most likely as a consequence of nicotine-promoted nAChR desensitization.4. Augmented ACh release and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in other neurons, increases intracellular Ca²⁺ (Cholewinskiet al., 1993), thus promoting desensitization of ACh release.     

Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

Nicotine trapping causes the persistent desensitization of alpha4beta2 nicotinic receptors expressed in oocytes

To determine whether prolonged nicotine exposure persistently inactivates rat alpha4beta2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped alpha4beta2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 microm nicotine. A 12-h incubation in 10 microm nicotine depressed the alpha4beta2 ACh response for 24 h without affecting total or surface alpha4beta2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an alpha4beta2-expressing oocyte with an un-injected one (pre-incubated in 10 microm nicotine for 12 h) for 24 h and measured the change in the alpha4beta2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 microm nicotine and a 24-h incubation in nicotine-free media. Thus, oocyte-mediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 microm nicotine. Consistent with this result, measurements of [3H]nicotine release show that oocytes release enough nicotine into the wash media to desensitize alpha4beta2 receptors and that prolonged incubation in 300 microm ACh (which cannot readily cross the membrane or accumulate in acidic vesicles) did not persistently depress the alpha4beta2 response.     

Evolutionary analysis of the carnitine- and choline acyltransferases suggests distinct evolution of CPT2 versus CPT1 and related variants

Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensive phylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.     

Nicotine-induced up-regulation and desensitization of alpha4beta2 neuronal nicotinic receptors depend on subunit ratio

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.