ACh-induced Desensitization Research Articles

Inhibition of oxotremorine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

The aim of this study was to investigate the differences between oxotremorine-induced and acetylcholine (ACh)-induced desensitization, particularly under Ca2+-free conditions, in guinea-pig ileal longitudinal muscle, and to elucidate the different mechanisms of desensitization that might exist between these two muscarinic agonists. Pretreatment of the tissue with 10(-7)-10(-5) M oxotremorine (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses to ACh, as did the desensitizing treatment with ACh. However, Ca2+-free conditions significantly reduced oxotremorine-induced desensitization, contrary to the previous findings that Ca2+-free conditions enhanced ACh-induced desensitization. The desensitizing treatment with oxotremorine caused suppression of the responses to high K+ (tonic phase), as did the ACh treatment. Ca2+-free conditions removed this suppression, whereasthis condition enhanced ACh-induced suppression of the K+ response. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10(-4) M) had no effect on oxotremorine-induced desensitization of the ACh response. The results suggest that a voltage-gated Ca2+ channel was involved in oxotremorine-induced desensitization, as in ACh-induced desensitization, but that the process of inactivation of Ca2+ channels was different between oxotremorine and ACh, and that oxotremorine-induced desensitization was due not only to Ca2+ channel, but also to other unknown factors. Protein kinase C did not participate in oxotremorine-induced desensitization.

The local anaesthetics proadifen and adiphenine inhibit nicotinic receptors by different molecular mechanisms

Many local anaesthetics are non-competitive inhibitors of nicotinic receptors (acetylcholine receptor, AChR). Proadifen induces a high-affinity state of the receptor, but its mechanism of action and that of an analogue, adiphenine, is unknown. We measured the effects of proadifen and adiphenine on single-channel and macroscopic currents of adult mouse muscle AChR (wild-type and mutant). We assessed the results in terms of mechanisms and sites of action. Both proadifen and adiphenine decreased the frequency of ACh-induced single-channel currents. Proadifen did not change cluster properties, but adiphenine decreased cluster duration (36-fold at 100 micromolxL(-1)). Preincubation with proadifen decreased the amplitude (IC(50)= 19 micromolxL(-1)) without changing the decay rate of macroscopic currents. In contrast, adiphenine did not change amplitude but increased the decay rate (IC(50)= 15 micromolxL(-1)). Kinetic measurements demonstrate that proadifen acts on the resting state to induce a desensitized state whose kinetics of recovery resemble those of ACh-induced desensitization. Adiphenine accelerates desensitization from the open state, but previous application of the drug to resting receptors is required. Both drugs stabilize desensitized states, as evidenced by the decrease in the number of clusters elicited by high ACh concentrations. The inhibition by adiphenine is not affected by proadifen, and the mutation alphaE262K decreases the sensitivity of the AChR only for adiphenine, indicating that these drugs act at different sites. Two analogous local anaesthetics bind to different sites and inhibit AChR activity via different mechanisms and conformational states. These results provide new information on drug modulation of AChR.

Kinetics of desensitization and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells.

Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Whole-cell patch clamp recordings were performed to evaluate alpha4beta2-nAChR currents. Application of 0.1 micromol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of approximately 70 and approximately 700 ms, for the onset of alpha4beta2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of alpha4beta2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs(1) from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human alpha4beta2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings(2) using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. These findings indicate the existence of alpha4beta2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.

Photoactivated 3-Azioctanol Irreversibly Desensitizes Muscle Nicotinic ACh Receptors via Interactions at αE262

3-Azioctanol is a photoactivatable analogue of octanol that noncompetitively inhibits nicotinic acetylcholine receptors (nAChRs). Photolabeling studies using [3H]-3-azioctanol in Torpedo nAChR identified alphaE262 as a site of desensitization-dependent incorporation. However, it is unknown whether photolabeling of alphaE262 causes functional effects in nAChRs and what other roles this residue plays in gating, desensitization, and channel block. We used ultrafast patch-perfusion electrophysiology and ultraviolet (UV) irradiation to investigate the state-dependence of both reversible nAChR inhibition by 3-azioctanol and the irreversible effects of photoactivated 3-azioctanol. Channels with mutations at alphaE262 were studied to determine ACh EC50s, desensitization rates, and sensitivities to reversible and photoirreversible 3-azioctanol inhibition. Exposure to 3-azioctanol in the presence of 365 nm UV light produced irreversible inhibition of wild-type nAChRs. Desensitization with ACh dramatically increased the degree of irreversible inhibition by photoactivated 3-azioctanol. Mutations at alphaE262 that reduce diazirine photomodification decreased the irreversible inhibition induced by photoactivated 3-azioctanol. Hydrophobic mutations at alphaE262 significantly slowed rapid ACh-induced desensitization and dramatically slowed fast resensitization. In contrast, alphaE262 mutations minimally affected 3-azioctanol channel block, and a half blocking concentration of 3-azioctanol did not alter the rate of ACh-induced fast desensitization. Our results indicate that position alphaE262 on muscle nAChRs contributes to an allosteric modulator site that is strongly coupled to desensitization. Occupation of this pocket by hydrophobic molecules stabilizes a desensitized state by slowing resensitization.

Subunit-Dependent Modulation of Neuronal Nicotinic Receptors by Zinc

We examined the effect of zinc on rat neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes as simple heteromers of alpha2, alpha3, or alpha4 and beta2 or beta4. Coapplication of zinc with low concentrations of acetylcholine (</=EC(10)) resulted in differential effects depending on receptor subunit composition. The alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, and alpha4beta4 receptors exhibited biphasic modulation by zinc, with potentiation of the acetylcholine response occurring at 1-100 micrometer zinc and inhibition occurring at higher zinc concentrations. In contrast, alpha3beta2 receptors were only inhibited by zinc (IC(50) = 97 +/- 16 micrometer). The greatest potentiating effect of zinc was seen with alpha4beta4 receptors that were potentiated to 560 +/- 17% of the response to ACh alone, with an EC(50) of 22 +/- 4 micrometer zinc. Cadmium, but not nickel, was also able to potentiate alpha4beta4 receptors. Both zinc potentiation of alpha4beta4 receptors and zinc inhibition of alpha3beta2 receptors were voltage independent. The sensitivity of zinc potentiation of alpha4beta4 to diethylpyrocarbonate treatment and alterations in pH suggested the involvement of histidine residues. Zinc continued to inhibit alpha4beta4 and alpha3beta2 after diethylpyrocarbonate treatment. Application of a potentiating zinc concentration increased the response of alpha4beta2 and alpha4beta4 receptors to saturating ACh concentrations. The rate of Ach-induced desensitization of these receptors was unaffected by zinc. Our results reveal zinc potentiation as a new mode of neuronal nAChR modulation.

Phosphatidylinositol 4,5-Bisphosphate Is Acting as a Signal Molecule in α1-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).

Enhancement of acetylcholine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

1. To determine the role of cellular Ca2+ in desensitization, acetylcholine(ACh)-induced desensitization was studied under Ca2+-free condition in guinea-pig ileal longitudinal muscle. 2. Pretreatment of the tissue with 10(-4) M ACh (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses both to ACh and histamine. The desensitizing treatment performed in Ca2+-free solution enhanced desensitization of the responses to ACh and histamine significantly. 3. The desensitizing treatment with ACh caused suppression of the responses to high K+ (tonic component) and Bay K 8644. The desensitizing treatment performed in Ca2+-free solution potentiated the suppression of the responses to high K+ and Bay K 8644 significantly. 4. ACh-induced desensitization was enhanced significantly in the presence of a protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7, 10(-4) M) to a similar extent as desensitization obtained under Ca2+-free condition, but not in the presence of a non-specific and less potent kinase inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004, 10(-4) M). 5. These results suggested that voltage-gated Ca2+ channels were involved in ACh-induced desensitization and that intracellular Ca2+, which was increased during the stimulation with ACh, inhibited desensitization through the activation of protein kinase C. This kinase could have activated or protected Ca2+ channels during the desensitization process to reduce desensitization.

Peptide modulation of ACh receptor desensitization controls neurotransmitter release from chicken sympathetic neurons.

1. The distribution and release of substance P (SP) in embryonic chicken lumbar sympathetic ganglia was examined with the use of immunohistochemistry and radioimmunoassay, respectively. SP immunoreactivity was detected in nerve fibers surrounding individual sympathetic neurons and was released by ganglionic depolarization. 2. Effects of SP on nicotinic acetylcholine receptor (AChR) function was assayed in embryonic sympathetic neurons in vitro by whole-cell patch clamp. SP (0.1-20 microM) accelerated the rate of decay (desensitization) of ACh-induced currents. The AChR desensitization time course is biphasic and described by the sum of two exponential functions dependent on agonist concentration (time constant of the faster component, tau f = 1-2 s, and the slower time constant, tau s = 10-25 s). SP selectively decreased tau s and the contribution of the slow component to the overall rate of current decay. The effects of SP on desensitization were concentration dependent and reversible. SP slowed recovery from desensitization by 2.5-fold. 3. SP shifted the dose-response curve for ACh-induced desensitization, reducing the concentration of ACh required to produce half-maximal desensitization by approximately twofold. 4. Preapplication of SP was equivalent to SP applied together with ACh in accelerating AChR desensitization. SP did not alter the time course of currents elicited by nondesensitizing concentrations of ACh, carbamylcholine (CARB), or dimethylphenylpiperazinium (DMPP). These data suggest that AChR activation is neither necessary nor sufficient for the peptide to modulate receptor function. A kinetic model of the effects of SP on specific steps in AChR desensitization is presented. 5. SP enhanced the rate of decay of synaptic currents in sympathetic neurons innervated in vitro, decreasing the synaptic current duration by up to 80%. 6. Effects of SP on neurotransmitter release from sympathetic neurons were evaluated by measuring the release of [3H]-norepinephrine (NE). ACh and CARB stimulated NE release in a concentration- and calcium-dependent manner. SP alone had no effect on NE secretion, but the peptide inhibited NE release induced by ACh or CARB by 40-50%. 7. Although agonists specific for either nicotinic or muscarinic receptors stimulated release of NE, SP selectively inhibited the nicotinic component of transmitter secretion. Thus SP suppressed NE release induced by DMPP by up to 80% but had no effect on muscarine or depolarization-induced NE secretion. 8. Parallel studies of the modulatory effects of SP on whole-cell currents and NE secretion revealed that SP inhibition of transmitter release from sympathetic neurons is directly proportional to the extent of potentiation of AChR desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Discriminative stimulus properties of nicotine: mechanisms of transduction.

Our thinking about how nicotine might be inducing DS control of behavior has changed drastically in the past 25 years. Our first inclination was that nicotine was mimicking ACh at a variety of specific and select n-AChRs. Then several nicotine researchers suggested that nicotine might be acting via specific and select noncholinergic receptors. At present, we seem to have returned to the view that nicotine may have pronounced effects at the n-AChR (figure 1), at least in some specific cases such as in the development of tolerance and in rats trained to discriminate nicotine (figure 6). The endogenous ligand has not changed, but the mechanism of how it affects presynaptic and postsynaptic receptors appears to have been rediscovered. The concept of rapid ACh-induced desensitization at the n-AChR is not new and appears basic to cholinergic neuronal function (figure 5). The desensitization concept has been revitalized by several investigators who also consider this mechanism important to how nicotine might act in protecting DA neurons from neurotoxicity of chemicals such as 6-OHDA or MPTP (Janson et al. 1988). The overall concept suggests that n-AChR desensitization at presynaptic DA sites may reduce neuronal accessibility to select exogenous neurotoxins and thus attenuate neuronal destruction. This hypothesis has also been partially validated in relation to the cholinergic neuron by providing preliminary evidence that nicotine was able to reduce cholinergic neuron destruction (measured by brain ACh levels) via the intraventricular administration of the neurotoxin AF64A, an ACh nitrogen mustard (Villanueva et al. 1990). Thus, nicotine or compounds acting like nicotine could possibly be beneficial to patients exhibiting the select neurological problems observed in Parkinson's and Alzheimer's diseases. The process of desensitization might also be useful to understanding why humans choose to smoke tobacco products. Perhaps the ability of nicotine to induce such neuronal effects at a specific n-AChR makes it reinforcing (or aversive) to behavior. Such a mechanism of action might explain why nicotine appears to both increase and decrease arousal levels in animals (or humans) exhibiting differential basal level of excitability (Hendry and Rosecrans 1982), or why some people never become dependent on nicotine. How nicotine alters the n-AChR may also be beneficial to our understanding of the subtle nature of cholinergic neuronal function in learning, memory, and other behavioral states. The ability of ACh (or nicotine) to induce an activation or attenuation of some cholinergic or noncholinergic neuron or both may be important to these brain processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of calcium in acetylcholine-induced desensitization in dog thyroid slices

Incubation of dog thyroid slices with 1 μ m acetylcholine (ACH) for 3 h followed by a second 2-h incubation without it induces a diminution of stimulation of glucose oxidation by ACH during a third incubation of 45 min. Using a calcium-free medium during all incubations prevents the desensitization and reduces, but does not abolish, ACH stimulation of glucose oxidation. EGTA [ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid] (2 m m) added to the calcium-free medium in all incubations prevents both refractoriness and stimulation of glucose oxidation induced by ACH. Calcium depletion during the first incubation only, achieved by using EGTA and a calcium-free medium, also prevents refractoriness but not the augmentation of glucose oxidation caused by ACH. Incubation of thyroid slices with 1 μ m ionophore A23817 during the 3-h first incubation decreases the stimulation of glucose oxidation induced by its readdition or by 1 μ m ACH added for the first time in the third incubation. Ionophore-induced desensitization is not related to a cholinergic muscarinic receptor effect. Initial incubation of dog thyroid slices with 1 μ m ACH diminishes the subsequent stimulation of glucose oxidation by 0.5 μ m ionophore. However, the ACH-induced desensitization to ionophore can be overcome by a 10-fold increase in the amount of ionophore in the third incubation. Ionophore (1 μ m) in the first incubation also induces refractoriness to thyroid-stimulating hormone (TSH) (10 mU/ml)-stimulated glucose oxidation in the third incubation. In contrast, initial incubation of thyroid slices with TSH (25 mU/ml) does not affect the stimulation of glucose oxidation by 0.5 μ m ionophore added during the third incubation. These results suggest that increased intracellular calcium plays a major role in, or even mediates, ACH-induced desensitization in the thyroid gland.