Role of local anesthetics on both cholinergic and serotonergic ionotropic receptors

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Abstract

A great body of experimental evidence indicates that the main target for the pharmacological action of local anesthetics (LAs) is the voltage-gated Na+ channel. However, the epidural and spinal anesthesia as well as the behavioral effects of LAs cannot be explained exclusively by its inhibitory effect on the voltage-gated Na+ channel. Thus, the involvement of other ion channel receptors has been suggested. Particularly, two members of the neurotransmitter-gated ion channel receptor superfamily, the nicotinic acetylcholine receptor (AChR) and the 5-hydroxytryptamine receptor (5-HT3R type). In this regard, the aim of this review is to explain and delineate the mechanism by which LAs inhibit both ionotropic receptors from peripheral and central nervous systems. Local anesthetics inhibit the ion channel activity of both muscle- and neuronal-type AChRs in a noncompetitive fashion. Additionally, LAs inhibit the 5-HT3R by competing with the serotonergic agonist binding sites. The noncompetitive inhibitory action of LAs on the AChR is ascribed to two possible blocking mechanisms. An open-channel-blocking mechanism where the drug binds to the open channel and/or an allosteric mechanism where LAs bind to closed channels. The open-channel-blocking mechanism is in accord with the existence of high-affinity LA binding sites located in the ion channel. The allosteric mechanism seems to be physiologically more relevant than the open-channel-blocking mechanism. The inhibitory property of LAs is also elicited by binding to several low-affinity sites positioned at the lipid–AChR interface. However, there is no clearcut evidence indicating whether these sites are located at either the annular or the nonannular lipid domain.

Both tertiary (protonated) and quaternary LAs gain the interior of the channel through the hydrophilic pathway formed by the extracellular ion channel's mouth with the concomitant ion flux blockade. Nevertheless, an alternative mode of action is proposed for both deprotonated tertiary and permanently-uncharged LAs: they may pass from the lipid membrane core to the lumen of the ion channel through a hydrophobic pathway. Perhaps this hydrophobic pathway is structurally related to the nonannular lipid domain.

Regarding the LA binding site location on the 5-HT3R, at least two amino acids have been involved. Glutamic acid at position 106 which is located in a residue sequence homologous to loop A from the principal component of the binding site for cholinergic agonists and competitive antagonists, and Trp67 which is positioned in a stretch of amino acids homologous to loop F from the complementary component of the cholinergic ligand binding site.

Introduction

The concept of local anesthesia started to be acknowledged as early as 1884 when the natural alkaloid (−)cocaine, obtained from the leaves of the coca shrub Erythroxylon coca, was introduced into clinical practice (reviewed in Ref. [1]). However, due to its adverse secondary effects (toxicity and addictive properties), the search for alternative substitutes started eight years later. Soon after, the synthesis of procaine, the prototype for local anesthetics (LAs), was successfully achieved in 1905. The experience gained from these first observations paved the way for the actual medical use of LAs.

The most important pharmacological property of LAs is that they act on any nervous fiber from the nervous system (central or peripheral) inhibiting the action potentials responsible for nerve conduction. For instance, the topical application of certain LAs in a sensitive tissue (e.g. eyes, mucus membranes, or skin) causes loss of sensation with the subsequent mitigation of pain. In the neuromuscular junction, LAs cause motor paralysis in the treated innervated area. In the central nervous system, LAs produce an apparent stimulation and subsequent depression. These effects are reversible at clinically relevant concentrations: after a certain time, the nerve function is completely recovered without damage of the nerve fiber or cell under treatment.

Despite the vast clinical use of LAs, the molecular basis for local anesthesia started to be understood only 20 years ago. The inhibitory action of LAs on nerve conduction is primarily due to the interaction of the drug with voltage-gated Na+ channels. Relevant aspects on voltage-gated Na+ channels such as their structures and their functions on both central and peripheral systems can be found in several excellent reviews [1], [2], [3]. Other experimental evidence supports the conjecture that LAs may also act on several membrane-embedded proteins such as different types of K+ and Ca2+ channels and ion pumps as well as cyclases, kinases, phospholipases, and calmoduline (reviewed in Ref. [1]; see also Ref. [4] and references therein).

Although Na+ channels are directly associated to the mechanism of local anesthesia in the peripheral nervous system, the process of spinal and epidural anesthesia, which cause sympathetic nervous system paralysis, is more complex and poorly understood (reviewed in Ref. [1]). Clinical observations indicate the existence of other receptive proteins as pharmacological targets, in addition to voltage-sensitive Na+ channels, for the action of LAs during spinal and epidural anesthesia [5]. For example, presynaptic Ca2+ channels have been involved in the inhibitory action of LAs on synaptic transmission in the spinal cord (reviewed in Ref. [2]). Inhibition of these channels reduces the amount of neurotransmitter released during depolarization thus, suggesting the neurotransmitter release inhibition as one of the mechanisms by which LAs exert their effects on spinal cord. In addition, several postsynaptic receptors, including the tachykinin type 1 [6] and the nicotinic acetylcholine (AChR) receptors have been related to the LA-induced spinal cord anesthesia (reviewed in Ref. [2]).

The synaptic transmission in sympathetic ganglia can also be blocked by LAs. One proposed mechanism is that LAs decrease and eventually eliminate excitatory postsynaptic potentials [7]. Among postsynaptic receptors, the AChRs is one of the best studied receptor family that mediates excitatory chemical transmission at both peripheral (ganglionic and neuromuscular) and central synapses. In this regard, LAs inhibit both muscle- and neuronal-type AChRs in a noncompetitive fashion. Interestingly, LAs also block the excitatory response of the ionotropic receptor for the neurotransmitter 5-hydroxytryptamine (5-HT), the 5-HT3R type [3], [4], [5], [6], [7], [8], [9], [10], [11] (reviewed in Ref. [12]). However, the observed inhibition is considered to be mediated by a mechanism that differs from that addressed in both the voltage-gated Na+ and the AChR channel. Likewise, the elicited LA inhibition of the 5-HT3R may play a role in the mechanism of local anesthesia since the activation of this receptor mediates pain sensation in peripheral tissues (reviewed in Ref. [13]). In addition, the activation of the 5-HT3R mediates other kinds of pain such as maigraine, angine, irritable bowel syndrome, and nociceptive responses to intravenous administration of 5-HT (reviewed in Ref. [14]).

Finally, procaine and other LAs induce some behavioral effects that cannot be solely explained by its action on voltage-gated Na+ channels (reviewed in Ref. [15]). Thus, it is clear that the pharmacological effects of LAs are broader than simple pain-relieving. Considering that both ionotropic receptors are involved in several higher-order brain functions such as cognition and behavior, the LA-induced behavioral effect may include ionotropic receptors, particularly neuronal AChR and 5-HT3Rs.

Within this perspective, a review addressing the updated information for the molecular basis of the inhibitory action of LAs on both muscle- and neuronal-type AChR ion channels and its comparison with the mechanism of 5-HT3R inhibition will be of theoretical and practical interest for general readers as well as for those researchers who are involved in structural and functional studies on ligand-gated ion channels. No less important is the fact that LAs have been efficiently used as probes to obtain structural information from AChR ion channels. For this purpose, we will briefly describe the basic structure and function of both ionotropic receptors. Then, we will explain structural aspects of LAs and the mechanisms by which they act on the AChR. And, finally, the localization of LA binding sites on both inotropic receptors and some of the behavioral effects of LAs on the central nervous system will be addressed. Pharmacokinetical and toxicological studies on LAs will not be considered in this review. For these issues, we suggest the reader to see the review by Catteral and Mackie [1].

Section snippets

Structural components of the nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors

Up to date, nine α (α1–α9), four β (β1–β4), one γ, one ε, and one δ subunit-encoding genes have been identified in several tissues and cells from different species (reviewed in [16], [17]). In addition, spliced forms of both γ [18] and α3 [19] subunits have been distinguished. Subunits of the same class (e.g. α subunits) from a given species present high homology (∼80%). Among distinct species the homology is lower (∼40%). In muscle cells, the subunits α1, β1, γ, ε, and δ have been found in the

Basic function of nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors

All observed functional properties of this receptor superfamily can be described in terms of four main attributes: (1) they have the ability to recognize and to bind its specific neurotransmitter; (2) upon ligand binding, the intrinsic ion channel associated with each particular receptor protein becomes opened; (3) after channel opening, specific ions (e.g. Cl for GlyR, GABAAR and GABACR, and Na+, K+ and Ca2+ for AChRs and the 5-HT3R) are selected and conducted through the lipid membrane; and

Structural features of local anesthetic molecules

The LAs can be chemically considered as aromatic amines (reviewed in Ref. [40]). The molecular structure of LAs contains both hydrophilic and hydrophobic portions which are separated by an intermediate ester or amide linkage. Local anesthetics with an ester bond can be readly hydrolyzed by esterases from the plasma. Depending on the number and type of aromatic rings, three main groups can be distinguished (see molecular structures in Fig. 2).

  • Group I: LAs that bear only one aromatic ring.

Molecular mechanisms of local anesthetic inhibition of the AChR

The early use of several electrophysiological techniques such as voltage jump relaxation and agonist-induced noise spectra analysis (e.g. see [42], [43], [44], [45]) demonstrated that LAs depress synaptic transmission by inhibiting the AChR. These and subsequent studies on single agonist-activated ion channels from both muscle- [46], [47], [48], [49], [50], [51] and neuronal-type AChRs [7], [52], [53] and rapid 86Rb+ quenched-flux assays [54] supported the notion that the pharmacological action

Local anesthetic binding sites at the AChR

Experimental evidence for the localization of LA binding sites on the AChR was obtained by using a wide range of different techniques borrowed from the field of spectroscopy, electrophysiology, biochemistry, pharmacology, and molecular biology. For example, two main binding site classes for LAs have been proposed on the basis of pharmacological evidence [69]. (a) One class is formed by a population of 10–30 binding sites, probably located at the lipid–protein interface. These sites presents

Nonluminal local anesthetic binding sites

Binding experiments with [3H]trimethisoquin demonstrated that the low-affinity site concentration increases whereas the high-affinity site concentration remains without change when the lipid content of AChR reconstituted systems is augmented [69]. More specifically, at phospholipid:AChR molar ratios of 172±14, 236±20, and 310±20, the low-affinity site concentrations were 27±5, 40±5, and 59±5 μM, respectively, whereas the high-affinity site concentrations were 0.9±0.3, 1.0±0.3, and 0.8±0.3 μM,

Luminal local anesthetic binding sites

The early idea of the existence of an unique LA site located in the lumen channel which upon LA binding sterically blocks the ion flux activity, has been challenged by several lines of experimental support. Like in the case of the study of the localization of other NCI binding sites (reviewed in [24], [56], [70]), one of the most important techniques used to discern the localization of high-affinity LA binding sites is the photolabeling approach. This can be achieved by using radiolabeled LAs

The hydrophobic pathway for uncharged local anesthetics

By studying the effect of agonists on the interaction of several LASLs with the AChR by EPR, interesting results were obtained. In the presence of CCh, several LASL derivatives showed a slightly lower affinity for the AChR (Table 7). The same basic results were obtained when AChR-containing membranes were preincubated with C6SL (or alternatively with C6SL–MeI) and then incubated with CCh or when both ligands were incubated together. In this latter procedure, a fraction of AChRs may be initially

Ionic strength and pH dependence of local anesthetic binding to the AChR

Depending on the pH of the medium, tertiary amine LAs may acquire a proton and a positive charge (reviewed in Ref. [40]). The amine group from different LAs has intrinsic pKs that range from pH 8 to 9. For LASLs the pK values are in the range of 7.2–8.1 (see Table 9). Thus, at the used pHs (7.0–8.0), the pharmacological effect of LAs is represented by the sum of the populations of uncharged and charged molecules within the membrane, which is dominated by the neutral species. The interaction of

Local anesthetic binding sites at the 5-HT3R

Regarding other members of the ligand-gated ionotropic receptor superfamily, the agonist-induced current in the 5-HT3R ion channel is 50% inhibited (IC50) by QX-222 and procaine at concentrations of 8.5 [11] and 1.7 μM [10], respectively (Table 10). Likewise, tetracaine and (−)cocaine also inhibited the 5-HT3R in the same concentration range [9]. These values are slightly lower than those found for the AChR noncompetitive inhibition (see Table 2). However, the values obtained from LA-induced

Behavioral effects of local anesthetics

As was outlined in the Introduction, LAs, in addition to block the nerve conduction in the peripheral nervous system, may affect the central nervous system as well. When LA molecules reach the central nervous system, e.g. after intravenous infusion and later absorption, an apparent stimulation and subsequent depression is observed (reviewed in Ref. [1]). During the stimulatory phase, symptoms such as restleness, euphoria, muscle switching, tremor, and clonic convultions have been noticed ([10]

Conclusions

A major focus of current research on the ligand-gated ionotropic receptor superfamily has been to understand the molecular mechanism of action of LAs. Experimental evidence for the existence and location of both luminal and nonluminal LA binding sites on the AChR has been addressed in this review. The simplest mechanism to describe the action of LAs which bind to luminal sites assumes that these compounds enter the open channel, bind to different rings within the M2 transmembrane AChR domain,

Acknowledgements

Part of this work was performed at the Structural Biology Laboratory, University of São Paulo, Brazil, thanks to the financial support of Fundação de Amparo á Pesquisa do Estado de São Paulo (FAPESP). The kind hospitality and discussions with Prof Dr Shirley Schreier are gratefully acknowledged. I also thank Prof Dr Ariel Fernández for his help with the English language and to Marcelo Distéfano for his help with the art work.

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