Tina K. Machu, Dept. of Pharmacology Texas Tech University Health Sciences Center

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Glycine receptors are the predominant inhibitory neurotransmitter receptors in brain stem and spinal cord (reviewed in Betz, 1991). Purified glycine receptors contain two homologous polypeptides, alpha and beta. To date, four alpha and one beta subunits have been identified through molecular cloning techniques. The ligand-binding site of the glycine receptor has been localized to the alpha subunits, which form homo-oligomeric receptors in heterologous expression systems, such as the Xenopus laevis oocyte (Grenningloh et al., 1990a). In addition to being an integral subunit of the native glycine receptor, the beta subunit appears to be the site through which gephyrin, a putative cytoskeletal docking protein, binds to localize the receptor in postsynaptic membranes of neurons (Kirsch and Betz, 1995).

Glycine receptors in adult brain and spinal cord are accepted as being composed of alpha and beta subunits, given their insensitivity to picrotoxin, which the beta subunit confers (Pribilla et al., 1992). In contrast, glycine receptors in developing rat brain are presumed to be homomeric alpha₂ subunit receptors due to their sensitivity to picrotoxin (Akaike and Kaneda, 1989; Mori-Okamoto and Tatsuno, 1985) and the predominance of the alpha₂ subunit in the early stages of development (Betz, 1991). Glycine receptors are members of the superfamily of ligand-gated ion channels, of which the nicotinic receptor is the prototype. Many of these receptors are targets for alcohol and anesthetic modulation (Harris et al., 1995; Franks and Lieb, 1994; Sanna and Harris, 1993) and because of their roles in regulating synaptic transmission, ligand-gated ion channels are prime candidates for producing the mechanism of action for both classes of drugs. Very little work has been done to characterize the sensitivity of glycine receptors to alcohols or anesthetics. In general, ethanol has been reported to enhance glycine-induced Cl- currents in chick spinal cord neurons (Celentano et al., 1988), rat brain synaptoneurosomes (Engblom and Akerman, 1991), and cultured mouse neurons (Aguayo and Pancetti, 1994). The volatile anesthetics, halothane and enflurane, stimulate glycine-induced currents in dissociated neurons (Wakamori et al., 1991), while isoflurane enhances alpha₂ glycine receptors expressed in HEK293 cells (Harrison et al., 1993).

While alcohols and anesthetics share many neurochemical features, recent work has indicated that protein domains may confer sensitivity to these drugs, and furthermore, that these sites in ligand-gated ion channels may, in fact, be distinct. For instance, ethanol and halothane both enhance 5-hydroxytryptamine3 receptor function, but halothane potentiates receptor function in the presence of saturating concentrations of ethanol (Machu and Harris, 1994). Subunit requirements observed for stimulation of GABA_a receptor function by subanesthetic concentrations of ethanol (Wafford et al., 1991) are not shared by enflurane (Mihic et al., 1994), isoflurane (Harrison et al., 1993) or the intravenous anesthetic, propofol (Jones et al., 1995). Furthermore, members of the kainate/AMPA (GluR 1-3) and kainate (GluR6) family of receptors are all inhibited by ethanol (Dildy-Mayfield and Harris, 1995), while GluR1-3 are inhibited and GluR6 is stimulated by volatile anesthetics (Harris et al., 1995), respectively.

The sensitivities of cloned glycine receptors to alcohols and anesthetics have not been studied. As a first step, I have examined the actions of ethanol, butanol, and halothane on glycine mediated Cl- currents in oocytes expressing alpha₁ or alpha₂ glycine receptors. The characterization of these compounds on defined channels will provide the framework for further study of primary and secondary structural requirements for drug action.

Xenopus laevis female frogs were obtained from Xenopus I (Ann Arbor, MI). Glycine was purchased from Bio-Rad (Hercules, CA) and strychnine was obtained from Sigma (St. Louis, MO). Halothane was purchased from Ayerst (New York, NY). The bath concentration of halothane, 25% of its original bottle concentration, was determined by gas chromatography as previously described by Lin et al. (1992). The alpha₁ and alpha₂ glycine receptor cDNAs were provided by Dr. Heinrich Betz.

Isolation of Xenopus oocytes. X. laevis frogs were kept in tanks in dechlorinated tap water at 19^o C on a 12-hour light/dark cycle and were fed a diet of mealworms and beef heart 3 times per week. Frogs were anesthetized by immersion in ice water for 45 min and were kept on ice during surgery. After surgical removal, ovarian lobes were placed in modified Barth's solution (MBS) containing (in mM) NaCl 88, KCl 1, N_aHCO₃ 2.4, HEPES 10, M_gSO₄ 0.82, Ca(NO₃)₂ 0.33, C_aCl₂ 0.91, (pH 7.5).

Before dissection, ovarian lobes were placed in hypertonic isolation media containing (in mM) NaCl 108, KCl 2, EDTA 1, and HEPES 10 (pH 7.5). Stage V and VI oocytes were dissected with fine surgical forceps as previously described (Dildy-Mayfield and Harris, 1992) and placed in MBS. Oocytes were treated with collagenase Type 1A (0.5 mg/ml) in (mM) NaCl 83, KCl 2, MgCl2 1 and HEPES 10 (pH 7.5) to remove the follicular cell layer, and rinsed in MBS.

Microinjection of oocytes with Glycine Receptor cDNA. Glycine receptor cDNAs were subcloned in the pCis vector which expresses well when injected into the oocyte nucleus. The "blind" method for nuclear injection described by Colman (1984) was used to inject 50 pg - 5 ng of glycine receptor cDNA dissolved in 30 nl of diethyl pyrocarbonate (DEPC)-treated water per oocyte. Oocytes were maintained at 18^oC in sterile filtered incubation media (MBS containing 10 mg/l streptomycin, 50 mg/l gentamicin, 10,000 U penicillin/l, 0.5 mM theophylline, and 2 mM sodium pyruvate). Incubation media was changed daily. Oocytes were recorded from day 1 through 7 following injection.

Electrophysiological Recording of Glycine Receptor Responses. Oocytes were perfused (2 ml/min) in a 100 u1 volume recording chamber with MBS via a roller pump (Cole Parmer Instrument, Co., Chicago, IL). Oocytes were impaled with 2 glass electrodes (1.2 mm outside diameter and 1 - 10 megohm resistance) filled with 3 M KCl. Oocytes were voltage clamped to -70 mV using a Warner Instruments Model OC-725A (Hamden, CT) oocyte clamp. Clamping currents were plotted on a strip chart recorder (Cole Parmer Instrument Co., Chicago, IL).

Glycine was dissolved in MBS and applied for 30 sec. Alcohols and anesthetics were coapplied with glycine. Alcohols were preapplied for 1 min prior to glycine plus alcohol application.

Data Analysis Prism Software (San Diego, CA) was used to determine EC50s and Hill coefficients. Prism or Instat (San Diego, CA) Software was used to perform one- or two- way analysis of variance (ANOVA) and Student's t-tests. One-way ANOVA was used, in some cases, to evaluate the effects of drug concentration on a construct. Two-way ANOVA was used to simultaneously examine the effects of drug concentration and receptor construct. The unpaired Student's t-test was used to examine any differences in the effects of a single concentration of drug on the two receptor constructs.

Calculation of MAC values. Concentrations of anesthetics in the oocyte perfusion chamber were converted to concentrations at 37^o C with the equation reported by Franks and Lieb (1993):

(Concentration at 22^o C)/(Concentration at 37^o C) = exp [(-4.08(37-22))/(273.15+22)]

Concentrations at 37^o C were converted to MAC by the equation:

Molar concentration at 37^o C = MAC x (saline/gas partition coefficient) x ((273/310)/22.4)

In recent years, the hypothesis that alcohols and anesthetics produce central nervous system depression through the disordering of membrane lipids has fallen into disfavor. While lipophilicity, in general, is a good indicator of alcohol/anesthetic potency (Meyer, 1901; Overton, 1896), it is clear that membrane disordering can be produced by compounds that do not produce intoxication or anesthesia (Buck et al., 1989). Furthermore, Franks and Lieb (1984) showed that anesthetics have potent actions on purified proteins in the absence of any lipids. Their groundbreaking work with firefly luciferase gave rise to the hypothesis that alcohols and anesthetics may act through partitioning into hydrophobic domains of proteins. Because of their roles in mediating synaptic transmission, ligand-gated ion channels are prime protein candidates for mediating the behavioral sequelae associated with intoxicant-anesthetic drugs.

Numerous studies have been conducted on most of the members of the superfamily of ligand-gated ion channels, as well as the glutamate family of receptors, with respect to their modulation by alcohols (reviewed in Sanna and Harris, 1993) and anesthetics (reviewed by Harris et al., 1995). However, scant attention has been given to the strychnine sensitive glycine receptor, which is important in regulating motor and sensory functions (reviewed by Betz, 1991). While sparsely present in higher brain structures, the glycine receptor is plentiful in brainstem and spinal cord, two areas of the central nervous system which have been largely overlooked in recent years with regard to the pharmacology of alcohols and anesthetics. Two lines of evidence indicate that anesthesia may be produced by subcortical structures. In goats, the MAC for halothane is ~ 2.5 fold greater when midbrain and forebrain structures alone are administered drug than when the entire body is perfused (Antognini and Schwartz, 1993). MAC is unaltered in rats by decerebration (Rampil et al., 1993). While many of the behaviors associated with ethanol intoxication may likely be due to impairment of higher brain structures, motor incoordination could be, in part, attributable to ethanol actions at brain stem and spinal cord. Recent findings have shown that glycine administered intracerebroventricularly enhances the depressant properties of a hypnotic dose of ethanol as measured by loss of righting reflex (Williams et al., 1995). This enhanced depressant effect was antagonized by low doses of strychnine, suggesting that it occurred through the inhibitory glycine receptor and not through the glycine sensitive site of the NMDA receptor.

The stimulation of alpha₁ and alpha₂ glycine receptors by ethanol and butanol that I report here represents the first demonstration that the cloned glycine receptor is modulated by alcohols. Previous work by other investigators in spinal cord neurons (Celanto et al., 1988), rat brain synaptoneurosomes (Engleblom and Akerman, 1991), and cultured mouse neurons (Aguayo and Pancetti, 1994) has shown that native glycine receptors are stimulated by ethanol. Aguayo and Pancetti (1994) found that ethanol enhancement of glycine receptors in spinal neurons was not saturable, even at high ethanol concentrations (400 mM), which is similar to my finding of a lack of saturation with 200 mM ethanol. Similarly, I did not see saturation with butanol over the range of concentrations tested. To be consistent with a "hydrophobic pocket" hypothesis in which there are a finite number of sensitive site(s) in the receptor, there theoretically must be a concentration high enough to saturate the sites. However, in many cases, this concentration may not be water soluble, as we have observed with long chain alcohols (Mascia et al., submitted). The observation that butanol is more potent than ethanol is consistent with the hydrophobic pocket hypothesis, given that butanol, a more lipophilic alcohol than ethanol, would be predicted to partition into alcohol sensitive sites with higher affinity. In agreement with the other investigators cited above, I observed that ethanol had actions on glycine receptors in the intoxicating range (25-50 mM), as well as the anesthetic range (>150 mM), suggesting that glycine receptors could participate in the mechanism of action of both ethanol induced intoxication and anesthesia.

My finding that the function of alpha₁ and alpha₂ subunits of the glycine receptor is enhanced by the volatile anesthetic, halothane, is consistent with the hypothesis that general anesthesia may be entirely, or in part, mediated by an increase in inhibitory neurotransmission, as has been reported for the GABA_a receptor (reviewed by Harris et al., 1995). Only one other group has investigated glycine receptor subunit subtypes' sensitivities to anesthetics. Harrison and coworkers (1993) reported that alpha₂ glycine receptors expressed in HEK293 cells were stimulated by 0.82 mM isoflurane. Most evidence that glycine receptors are targets of anesthetics has been obtained in nervous tissue preparations. Wakamori and coworkers (1991) examined the actions of halothane and enflurane on glycine induced currents in neurons dissociated from ratnucleus tractus solitarius. Their studies showed that both volatile anesthetics enhanced glycine receptor function in a concentration dependent manner. Furthermore, they reported that enflurane and halothane decreased the Kd for glycine, without having any effect on the maximum glycine response. These results are mirrored by mine, since I observed an increase in glycine potency, but not efficacy with both alpha₁ and alpha₂ glycine receptors. I also observed that halothane increased glycine receptor responses in a concentration dependent manner. My results, as well as that of Harrison et al. (1993) and Wakamori et al. (1991), suggest that these volatile anesthetics act at glycine receptors in a pharmacologically relevant range. Wakamori et al. (1991) reported an increase in glycine mediated currents with halothane concentrations as low as ~ 0.5 MAC and plateauing at ~ 4 MAC. I observed potentiation of both alpha₁ and alpha₂ receptors with halothane concentrations as low as 0.25 MAC, but I did not observe saturation at the highest concentration (10 MAC) tested. These results all suggest that glycine receptors are potentiated by concentrations of halothane within the range of that used clinically (0.75-1.25 MAC), thus underscoring the possibility that the glycine receptor plays a role in the mechanism of action of these anesthetics.

A large number of classes of compounds produce anesthesia, some of which are fairly selective to one type of ligand-gated ion channel . For instance, the steroid alphaxalone primarily modulates GABA_a receptors (Hill-Venning et al., 1993), and the barbiturates are much more potent at GABA_a receptors than at other receptors. In the glutamate family of receptors, NMDA receptors are selectively inhibited by the dissociative anesthetic, ketamine (Macdonald et al., 1989; Macdonald et al., 1987). These observations suggest that, in some cases, specific drugs may act at one receptor type to produce anesthesia. However, other classes of anesthetics, such as the volatile anesthetics, are less discriminating and may produce anesthesia by interacting at multiple ligand-gated ion channels. These observations are consistent with the idea that anesthesia may be the sum total of stimulation of inhibitory neurotransmission and/or depression of excitatory neurotransmission that is specific to the respective class of anesthetic in question. Thus, the fact that glycine receptors are not responsive to barbiturates (Hales and Lambert, 1991) or alphaxalone (Hill-Vening et al., 1993), for instance, does not preclude their participation in mediating anesthesia produced by other classes of compounds such as the volatile anesthetics.

Stimulation by alcohols and anesthetics of homomeric alpha₁ and alpha₂ subunits of the glycine receptor expressed in oocytes reveals that a minimal requirement for such action in a glycine receptor is an alpha subunit. Glycine receptors present in adult brain or spinal cord are generally accepted to be composed of alpha and beta subunits. The premise for this notion is that native glycine receptors are insensitive to picrotoxin, whereas alpha subunit homomers are picrotoxin sensitive; the beta subunit confers picrotoxin insensitivity (Pribilla et al., 1992). However, in developing rat brain, alpha₂ subunits predominate (Betz, 1991), and these are presumed to be homomeric receptors, given the picrotoxin sensitivity of glycine receptors in developing rat brain (Akaike and Kaneda, 1989; Mori-Okamoto and Tatsuno, 1985). The results of the present study, taken together with previous work in nervous tissue preparations (Aguayo and Pancetti, 1994; Engleblom and Akerman, 1991; Wakamori et al., 1991; Celento et al., 1988), suggest that the presence of the beta subunit does not alter the sensitivity of glycine receptors to alcohols or anesthetics. Beta subunits are not very efficiently assembled with alpha subunits in expression systems, such as the Xenopus oocyte (Grenningloh et al., 1990b), and therefore were not tested. Thus the contribution, if any, of beta subunits to alcohol or anesthetic action glycine receptors is unknown. Beta subunits are more widely distributed in the CNS and in greater numbers than are alpha subunits (Betz, 1991), Beta subunit homomers in expression systems are weakly responsive to glycine in comparison with alpha subunits or native alpha plus beta subunits (Grenningloh et al., 1990b); therefore a direct contribution of beta subunit homomeric receptors, if they do occur in vivo, to glycine mediated neuronal inhibition via an increase in Cl- influx is doubtful. However, beta subunits appear to perform an important function in binding the glycine receptor to gephyrin, a cytoskeletal anchoring protein (Kirsch and Betz, 1995).

The nature of the site or sites in which alcohols and anesthetics act in glycine receptors, as well as other ligand-gated ion channels, remains unknown. Subunit specific requirements in recombinant GABA_a (Wafford et al., 1991) and kainate receptors (Harris et al., 1995) for alcohol/anesthetic modulation suggest a protein site of action. However, these sites may not be the same, given the lack of subunit specificity in the GABA_a receptor for potentiation by anesthetics (Jones et al., 1995; Mihic et al., 1994) versus the required presence of the PKC site of the gamma2L subunit of the GABA_a receptor for ethanol potentiation (Wafford et al., 1991). The requirement of the gamma2L insert of the GABA_a receptor for ethanol action remains controversial (reviewed by Macdonald, 1995). In any case, the ethanol induced stimulation that I report in the alpha₁ glycine receptor is not analogous to the GABA_a receptor, since I did not use the alpha₁ splice variant that contains a consensus sequence for PKC. However, other sites for protein kinase dependent phosphorylation in the glycine receptor have been described (Ruiz- Gomez et al., 1991), and these should be investigated for their potential role in the modulation of glycine receptor function by ethanol. My results with the 5-HT3 receptor suggest separate sites for ethanol and halothane, since halothane potentiates receptor function in the presence of a saturating concentration of ethanol (Machu and Harris, 1994). Similar studies were not done with the glycine receptor subtypes since saturating concentrations of either ethanol or halothane were not obtained. Thus, it remains unknown whether alcohols and anesthetics share a common site of action in glycine receptors.

In summary, I have demonstrated that cloned glycine receptors are targets for alcohol and anesthetic modulation. My work extends previous studies done in native nervous tissue preparations by showing that not only is an alpha subunit sufficient for modulation by these drugs, but also that there is no difference between the two alpha subunit subtypes tested. Glycine receptor subunits, like the 5-HT3 receptor, form homomeric receptors, which make them ideal candidates for testing amino acid requirements that confer sensitivity to alcohols or anesthetics.

Supported by funds from NIH grant AA10561.

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