Elsevier

Hearing Research

Volume 152, Issues 1–2, February 2001, Pages 25-42
Hearing Research

The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

https://doi.org/10.1016/S0378-5955(00)00225-2Get rights and content

Abstract

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between α9-nicotinic acetylcholine receptors (α9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of α9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells (∼90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with α9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the α9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the α9-nicotinic response, suggesting an enzymatic interaction with the α9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of α9nAChR, SK, or both.

Introduction

The impetus for investigating acetylcholine (ACh) and its receptors among inner ear sensory cells began over 40 years ago with the demonstration of acetylcholinesterase staining in cochlear and vestibular efferent fibers (Churchill et al., 1956, Dohlmann et al., 1958). A preponderance of evidence has since substantiated that ACh is indeed the major inner ear efferent transmitter among vertebrates (Eybalin, 1993, Guth et al., 1998). The frog offers a distinct advantage for studying the cholinergic function of vestibular efferents in that efferent fibers, after an extensive ramification at the level of Scarpa’s ganglion, are thought to innervate type II vestibular hair cells exclusively (Highstein, 1991, Precht, 1976, Lysakowski, 1996). Therefore, the effects elicited by efferent stimulation or ACh application on frog vestibular organs are most likely the result of ACh interacting with its receptors found on those hair cells. In frog vestibular organs, ACh has been shown to produce both facilitation and inhibition of afferent firing (Guth et al., 1986, Norris et al., 1988, Guth et al., 1994, Perin et al., 1998). The inhibition is thought to be mediated through the activation of α9-containing nicotinic receptors (α9nAChR) present on vestibular hair cells (Sugai et al., 1992, Elgoyhen et al., 1994, Yoshida et al., 1994, Hiel et al., 1996, Anderson et al., 1997, Athas et al., 1997a, Athas et al., 1997b, Gupta et al., 1997). Similar pharmacology has also been identified in auditory hair cells from many species (Housley and Ashmore, 1991, Shigemoto and Ohmori, 1991, Fuchs and Murrow, 1992a, Fuchs and Murrow, 1992b, Erostegui et al., 1994, McNiven et al., 1996, Nenov et al., 1996a). To date, all ACh-induced inhibition of afferent responses from both auditory and vestibular organs has been attributed to α9-containing nAChR activation and as such, the consequences of this activation also seem well conserved. Calcium influx via these receptors subsequently activates apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) resulting in an efflux of potassium, which subsequently hyperpolarizes the hair cell. In frog vestibular organs, this translates into a decrease in the release of transmitter by the hair cell with an ensuing reduction of afferent firing. To better examine the mechanisms underlying the α9nAChR-mediated effects on the level of vestibular hair cells, these cells can be enzymatically dissociated from select vestibular organs and examined individually using patch clamping methodology.

Cell dissociation techniques frequently involve enzymes in order to isolate individual cells from their respective tissues (Freshney, 1994). In this regard, sensory cells (hair cells) of the inner ear are no exception. The arsenal of enzymes commonly used to isolate vestibular hair cells has included papain and several serine proteases (Lewis and Hudspeth, 1983, Steinacker and Rojas, 1988, Housley et al., 1990, Holt and Eatock, 1995, Chabbert, 1997). Collagenase has been successfully used to isolate cells from the cochlea (Erostegui et al., 1994, Nenov et al., 1996a, Sugasawa et al., 1996, Nenov et al., 1998); but, it is rather ineffective in isolating frog vestibular hair cells. Initially, in this laboratory, a combination of papain and protease (VIII or XXIV) was successfully used for dissociating hair cells from frog vestibular organs. However, despite the detection of the α9nAChR subunit messenger RNA (mRNA) in isolated vestibular hair cells, we were unable to demonstrate ACh responses consistently in these cells. Failure to demonstrate routine ACh responses led to speculation that the α9nAChRs might undergo proteolysis during the isolation process and become non-functional. Indeed, a previous study had suggested that papain treatment might attenuate similar ACh responses in isolated hair cells from the turtle basilar papilla (Art and Goodman, 1996). Armstrong and Roberts (1998) have also recently shown that papain alters particular ion channels in frog saccular hair cells including SK, however, the exclusion of papain from our dissociation protocol did not significantly increase the number of ACh-responsive cells. It was therefore hypothesized that the remaining proteases (VIII or XXIV) might also participate in the degradation of components involved in the α9nAChR response in these cells.

Trypsin is also commonly used to isolate an assortment of different cell types. In many of these cells, receptors of interest have been shown to continue functioning following trypsinization (Freshney, 1994). Furthermore, trypsin has also been reported to affect membrane permeability and conductance only minimally (Narahashi and Tobias, 1964, Narahashi, 1974). When trypsin was substituted for either protease VIII or XXIV in the isolation protocol (papain excluded), the α9-nicotinic response to ACh was reliably and consistently observed in most (∼90%) of the saccular hair cells examined. In addition, ACh-produced responses were also identified in several solitary semicircular canal (SCC) and utricular hair cells isolated with trypsin. The pharmacology of the ACh response observed in isolated saccular hair cells was consistent with observations made in the intact organ as assessed by multiunit afferent firing recordings (reviewed in Guth et al., 1998). Moreover, trypsinized saccular hair cells demonstrated high intensity immunofluorescence when stained with anti-α9nAChR subunit antibody, providing an additional evidence for the presence of α9 subunit protein in these cells. A reliable response to ACh in trypsinized saccular hair cells provided the necessary means by which to investigate the effects of other enzymatic treatments on this response.

We report in this paper that both papain and two bacterial serine proteases (VIII, XXIV), enzymes commonly used to isolate vestibular hair cells, abolish the response to ACh mediated by α9-containing nicotinic receptors in these same cells. The α9-nicotinic ACh response, in both the intact organ and trypsinized saccular hair cells, was routinely and permanently eliminated by brief perfusions of either papain, protease VIII, or protease XXIV. The concentrations and exposure times mirror those used in previous isolation protocols. These same enzymes were without effect when inactivated by boiling before application. The abolition of the α9-nicotinic response in saccular hair cells is therefore most likely a function of the enzyme’s proteolytic activity. Similar perfusions with trypsin, collagenase, dispase, hyaluronidase, or elastase did not attenuate the α9-nicotinic response in frog saccular hair cells.

As evidenced here, when acutely isolated cells fail to behave as they do in situ, one of the problems may lie in the means used to obtain those cells. These observations explain the difficulty in demonstrating α9-nicotinic responses in solitary frog vestibular hair cells isolated with either papain, protease VIII, or protease XXIV. The novelty of this work is not necessarily that α9nAChRs are present in the frog saccule or that they underlie the inhibition of afferent discharge following efferent stimulation or exogenous ACh application. Instead, the contribution of this study is that the proteolytic enzymes that have been commonly used to isolate vestibular hair cells may alter or degrade some of the components that one may be studying. This is not an attempt to reevaluate previous work but to raise awareness about the deleterious effects that these enzymes may produce. Although receptor protein and its corresponding mRNA may be apparently preserved, the functional properties of the membrane’s molecules could be unpredictably changed following enzymatic treatment. The deleterious effects of these enzymes may have similar consequences for other isolation/dissociation protocols particularly using cells that might express similar membrane proteins including receptors and ion channels. Some of the results presented here have previously appeared in abstract form (Holt and Guth, 1999).

Section snippets

Multiunit afferent recordings

The method for recording the rate of saccular multiunit afferent firing was performed as previously described (Guth et al., 1994, Perin et al., 1998). The whole labyrinth bath (∼15 ml) was continuously superfused with artificial perilymph (AP) (in mM: 105 NaCl, 2.5 KCl, 0.81 MgCl2·6H2O, 1.8 CaCl2·2H20, 3.4 NaHCO3, 0.5 NaH2PO4·H2O, 2.5 Na2HPO4, 4 glucose) at a flow rate of 3–5 ml/min. Drugs were applied either by bath substitution or by close injection (15–45 s at 50 μl/min) through a

Multiunit afferent recordings

The application of 1 mM ACh routinely produces a robust inhibition of multiunit afferent firing as recorded from the intact frog saccule (Fig. 1A–D). This inhibition was potently blocked by strychnine (Fig. 1A) and attenuated by the classical nicotinic antagonists curare (Fig. 1B) and tetraethylammonium (Fig. 1C). Previous studies have demonstrated that strychnine (IC50=100 nM) was more potent than curare (IC50=400 nM) at blocking the α9nAChR-mediated response in the frog saccule (Guth et al.,

Discussion

We have demonstrated that the proteolytic enzymes papain, protease VIII, and protease XXIV permanently abolish the α9nAChR-mediated response in isolated frog saccular hair cells. It is suggested by the data that the complete and irreversible attenuation of the ACh response in these cells was the result of direct enzymatic alteration of components essential to the α9nAChR/SK mechanism.

There are several instances in the literature where enzymes have been shown to alter physiological responses.

Conclusions

A three-pronged approach using pharmacology, molecular biology, and immunocytochemistry supports the hypothesis that the inhibitory ACh response in isolated vestibular hair cells is most likely produced by an α9-containing nAChR. Furthermore, brief exposures to protease VIII, protease XXIV, and papain abolish the α9nAChR-mediated response in both the intact organ and in acutely isolated, trypsinized frog vestibular hair cells.

Acknowledgements

Work supported by NIH Grant DC-00303 (P.S.G.), DC-02364 (M.M.G.) and PhRMA (J.C.H.). The authors wish to thank Dr. Diane Blake for her timely advice, Cecilia E. Armstrong and Dr. William M. Roberts for their useful suggestions, and Dr. Robert J. Wenthold for the anti-α9 antibodies. We would also like to thank the Centralized Tulane Imaging Center (CTIC) for use of their fluorescent microscope and development of immunocytochemistry images. Special thanks is given to Amrita Puri, our dedicated

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