“… the role of GABA on airway epithelial and smooth muscle function and its possible role in airway pathology (e.g., asthma) is just beginning to be understood.”
IN this current issue of Anesthesiology, Xiang et al.1 describe the effects of volatile anesthetic activation of type A γ-amino butyric acid (GABAA) receptors expressed on airway epithelial cells. It has long been appreciated that a key mechanism in the action of general anesthetics in the brain involves the anesthetics’ allosteric potentiation of GABA on GABAA receptors, thereby augmenting this inhibitory neurotransmitter system. However, the demonstration that GABAA receptors are expressed extra-neuronally by a wide variety of cell types and modulate other important physiologic processes is a recent revelation. Although the bronchodilatory effects of volatile anesthetics have been clinically appreciated for many years, the signaling mechanisms by which this occurs in the lung are not fully known. The current study raises our awareness that anesthetics, via activation of GABAA receptors, may be having many effects on multiple cell types in the lung that not only mediate bronchodilation, but also effect immunomodulation and mucus production by epithelial cells. The emerging discovery of GABAA receptors on many peripheral cells raises important questions regarding the unrecognized effects of anesthetics on peripheral cellular processes.
The current study follows the seminal discovery (by these same authors) that multiple components of a GABAergic signaling system (GABAA receptors and the enzyme that synthesizes GABA [glutamic acid decarboxylase]) are expressed in airway epithelium and play an important role in the modulation of mucus production.2 In the current study, the authors focus on the alveolar type II epithelial (ATII) cells as they are also known to participate in immunomodulatory pulmonary processes (including specialized surfactant production and cytokine elaboration) which have important implications in inflammatory diseases of the lung such as asthma. With regard to the ATII cells, Xiang et al. advance our understanding of the GABAergic system in these cells by demonstrating the expression of multiple typical and atypical protein subunits of GABAA receptors collectively from human cultured alveolar epithelial type II (A549) cells as well as in situ human peripheral lung sections. Additionally, they elegantly demonstrate on these human cultured ATII cells classical currents in response to exogenous GABA with positive allosteric modulation by isoflurane and sevoflurane. Most interestingly, isoflurane impairs the basal expression of a key proinflammatory enzyme, cyclooxygenase-2, presumably via decreased calcium entry following GABAA receptor-mediated membrane depolarization. Opening of GABAA channels in ATII cells favors chloride efflux and depolarization in contrast to mature neurons where opening of GABAA channels favors chloride influx and membrane hyperpolarization due to differing electrochemical gradients in these cell types. These findings suggest a possible mechanism of anesthetics’ antiinflammatory effects not only in airway epithelial cells, but also on a host of other nonneuronal cells (including T lymphocytes) that have recently been found to express GABAA receptors.
It should be noted that the phenotype of GABAA subunits that Xiang et al. report on human ATII cells is consistent with subunit expression observed when GABAA receptors are found outside the neuronal synaptic cleft (i.e., “extrasynaptic GABAA receptors”). These extrasynaptic GABAA receptors within the central nervous system seem to be limited to only a small or restricted number of subunits that include α1,α4, α6 (in association with δ), or α5 (associating with either γ2 or δ).3 What is interesting is that in many nonneuronal tissues where GABAA receptors are found, this extrasynaptic phenotype and specialized subunit combination seem to be maintained. Xiang et al.’s current study shows in human cultured A549 cells that α5 and δ are present—which not only correlates well with the extrasynaptic phenotype, but also may explain the bidirectional electrophysiological effects of isoflurane that this group observed. These findings mirror the well-established observations that extrasynaptic GABAA receptors are responsive to lower concentrations of GABA and display slower desensitization rates when compared to GABAA receptors composed of subunits conforming to the more classical synaptic phenotype.4 Similarly, although unconventional, Xiang et al.’s observation that bicuculline could not completely reverse GABA-induced current is plausible, given published findings that shows competitive antagonists of the GABA binding site (such as bicuculline or gabazine) can exhibit differential blockade depending on GABAA receptor subunit composition.5,6 Furthermore, differential blockade of GABAA receptors is less established for the nonreversible antagonist picrotoxin, which agrees with Xiang et al.’s findings that this treatment abolished GABA-mediated current. This observation underscores an important point—that the role of the more “exotic” subunits (like the π subunit expressed in human ATII cells) may be modulating channel kinetics and pharmacological properties. This “theme” should be familiar to most anesthesiologists as pharmacologic specificity of benzodiazepine activation of GABAA receptors requires the inclusion of a γ subunit in the receptor. As such, current medicinal chemistry efforts are directed at designing ligands with selectivity for different α subunits of GABAA receptors that may allow selective targeting of specific neuronal or peripheral cell functions. However, caution is needed regarding potential subunit targeting of GABAA receptors in epithelial cells given the lack of potential correlation Xiang et al. observed between human and mouse ATII cells in lung sections and human A549 cells in culture. They demonstrate the GABAA α2 subunit in ATII cells in mouse lung sections but the GABAA α5 subunit in cultured human A549 cells. If there is indeed a lack of complete concordance of GABAA subunit expression in ATII cells between mice and human, continuing this work in another animal model where interspecies conservation is maintained, or (as was the case in this current article) working with human tissues and cells is essential to maintaining clinical relevance.
Perhaps the most intriguing component of this article is the potential link between GABAA receptors and inflammation. Xiang et al. demonstrate GABAA receptor-mediated reductions in the proinflammatory enzyme cyclooxygenase-2 in A549 cells treated with muscimol (a direct agonist of GABAA receptors) and isoflurane (allosteric agonist), and the reversal of this effect under conditions where isoflurane and bicuculline were coadministered. We applaud the authors for this seminal discovery, as it could be paradigm-shifting and establish another role for anesthetics beyond the central nervous system. However, while they certainly show reductions in constituitive expression of cyclooxygenase-2 (a key enzyme in arachidonic acid metabolism and prostanoid synthesis), it must be noted that the hallmark of this enzyme is that it is inducible and therefore exerts many of its proinflammatory cascades following an activating stimulus (such as lipopolysaccharide treatment).7 Although examining the effect of volatile anesthetics in attenuating cyclooxygenase-2 induction was beyond the scope of the current study, hopefully future research will address this important consideration.
Nevertheless, while one target of general anesthetics is GABAA receptors in the central nervous system, a host of other cell types may have altered signaling events due to simultaneous activation of their GABAA receptors with resulting changes in membrane potential and ion conductances. This may be particularly relevant in research related to the antiinflammatory effects and perhaps tumor surveillance aspects of leukocytes, a topic under intense clinical investigation with regard to cancer recurrence following exposure to opioids or local anesthetics. Perhaps the effects of activation of GABAA receptors on tumor surveillance cells by intravenous and inhaled anesthetics are an equally important area in need of further research.
The current study’s demonstration of an antiinflammatory role of isoflurane on airway epithelial cells follows a series of studies demonstrating a possible complex autocrine/paracrine signaling paradigm in the airway. Not only have GABAA receptors and the enzyme that synthesizes GABA, glutamic acid decarboxylase, been described in airway epithelium and airway smooth muscle,8 but GABA transporters type 2 and 4 have been shown to release GABA from airway epithelial cells9 and metabotropic (Gi-coupled) type-B GABA receptors have been described on airway epithelium, smooth muscle, and nerves. Thus, the role of GABA on airway epithelial and smooth muscle function and its possible role in airway pathology (e.g., asthma) are just beginning to be understood. Beyond the airway, the physiological effects of anesthetics on GABAA channels expressed on peripheral cell types (e.g., lymphocytes, renal tubule cells, and vascular and uterine smooth muscle) are largely unexplored and offer an opportunity for a new understanding of anesthetic effects on peripheral cells and organ function.