JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Streiff, J.
Right arrow Articles by Jones, K. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Streiff, J.
Right arrow Articles by Jones, K. A.
Related Collections
Right arrow Mechanisms
Right arrow Pharmacology

Anesth Analg 2004;98:660-7
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000099367.97415.5F

ANESTHETIC PHARMACOLOGY

The Effects of Hexanol on G{alpha}i Subunits of Heterotrimeric G Proteins

John Streiff, PhD*, David O. Warner, MD*, Elena Klimtchuk, PhD{dagger}, William J. Perkins, MD*, Kristofer Jones, BS{ddagger}, and Keith A. Jones, MD*

Departments of *Anesthesiology and {dagger}Physiology and Biophysics, {ddagger}Mayo Clinic and Mayo Foundation, Rochester, Minnesota

Address correspondence and reprint requests to David O. Warner, MD, Mayo Clinic and Foundation, 200 First St. S.W., Rochester, MN 55905. Address e-mail to warner.david{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Alcohols and other anesthetics interfere with the function of a variety of systems regulated by guanosine triphosphate (GTP)-binding proteins (G proteins). We examined the effect of hexanol on the activity of the {alpha} subunit (G{alpha}i1) of heterotrimeric G proteins. The GTP hydrolysis activity of recombinant G{alpha}i1 was 0.029 mole Pi · mole G{alpha}i1-1 · min-1 and was inhibited by hexanol at concentrations larger than 10 mM, with a 50% inhibitory concentration of 22 mM. Circular dichroism spectroscopy revealed that hexanol decreased the denaturation temperature of G{alpha}i1 from 47.2°C to 42.5°C without altering its secondary structure at 10°C. Hexanol (30 mM) reduced the amount of monomeric G{alpha}i1 in solution measured by size-exclusion chromatography, indicating that hexanol caused protein aggregation. However, the rate of GTP{gamma}S binding to G{alpha}i immunoprecipitated from airway smooth muscle membranes was not affected by 30 mM hexanol. Excluding the apparent inhibition of recombinant G{alpha}i1 resulting from aggregation-induced artifact, we found no evidence that the hexanol-induced inhibition of receptor-activated G{alpha}i-coupled pathways in intact airway smooth muscle resulted from direct inhibition of the intrinsic rate of [35S]GTP{gamma}S binding to G{alpha}i.

IMPLICATIONS: Although the {alpha} subunit of heterotrimeric G proteins is a potential target of anesthetics, we found no evidence that hexanol affects the ability of the G{alpha}i subunit to bind or hydrolyze guanosine triphosphate, either in purified subunits or in subunits derived from smooth muscle cell membranes. This finding implies that this is not a mechanism by which hexanol interferes with receptor-G protein function.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Alcohols have anesthetic properties in vivo and interact with isolated proteins in a manner similar to volatile anesthetics (1–3). The lower volatility and higher aqueous solubility of alcohols offer experimental advantages over volatile anesthetics. Our laboratory previously established that alcohols, like volatile anesthetics, relax airway smooth muscle (ASM). In particular, both hexanol and volatile anesthetics attenuate acetylcholine-induced increases in the amount of force produced for a given intracellular Ca2+ concentration (i.e., the calcium sensitivity) by inhibiting activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) coupled to muscarinic receptors (4–6). Studies in other systems also support the concept that alcohols and other anesthetics interfere with the coupling of receptors to G proteins. The mechanism of this effect is unclear, with evidence for association of anesthetics both with receptors (7) and G proteins (8). In support of the latter mechanism, Pentyala et al. (8) reported that volatile anesthetics inhibited the binding of [35S]guanosine triphosphate (GTP){gamma}S (a nonhydrolyzable analog of GTP) to {alpha} subunits of heterotrimeric G proteins (G{alpha}) in aqueous solution. This action should interfere with G-protein activation (GTP binding) and result in less guanosine triphosphatase (GTPase) activity of G{alpha}.

The original purpose of this investigation was to test the hypothesis that hexanol inhibits the [35S]GTP{gamma}S binding rate and GTPase activity of the heterotrimeric G-protein subunit G{alpha}i1. It became apparent that the effects of hexanol on the stability of the native recombinant G{alpha} required the use of a binding assay based on immunoprecipitation (IP) of subunit from ASM tissue homogenate. We also describe these destabilizing effects and how they can affect measurements of apparent anesthetic activity in vitro.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Hexahistidine-tagged recombinant G{alpha}i1 was generously provided by Alfred G. Gilman, MD, PhD. The GTPase activity of G{alpha}i1 was determined at 30°C over 20 min. Measurements were performed as described previously (9). Activity was determined for 200 nM G{alpha}i1 in 25 mM HEPES (pH 7.8), 100 mM NaCl, 10 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1 µM guanosine diphosphate (GDP), 5 µM GTP, 0.025 mCi [{gamma}32P]GTP, and 0.5 mg/mL bovine serum albumin with and without various concentrations of hexanol diluted from a stock solution. The concentration-response of G{alpha}i1-specific GTPase activity to hexanol was a nonlinear least-squares (NLS) fit with the equation y = max/(1 + e(x - IC50) x b), where max is the maximum specific activity, x is the hexanol concentration, IC50 is the inhibitor concentration yielding 50% inhibition, and b describes the slope of the line through the IC50 concentration.

Size-exclusion high-performance liquid chromatography (SEC-HPLC) was used to monitor aggregation of G{alpha}i1 in aqueous solution. SEC-HPLC separates molecules based on relative mobility through a porous media so that the largest molecules, which are excluded from the pores, elute first and the smaller molecules elute subsequently in order of size. Precolumn filtration prevents large particles from entering and potentially plugging the column. Size separation was performed on a BioSep-SEC-S 2000 PEEK column (Phenomenex, Torrance, CA) connected to an HPLC running 50 mM NaHEPES (pH 8) buffer at 1 mL/min. G{alpha}i1 (10 µg) was incubated in 210 µL of 50 mM NaHEPES (pH 8), 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, 5 µM GTP, and 0.1% polyoxyethylene 10 lauryl ether with various amounts of hexanol at 30°C. The G{alpha}i1 peak position was identified by its mass compared with molecular weight standards (Bio-Rad, Hercules, CA) run each day before measurements were performed. Injections (100 µL) of the assay mixture were made at 0 and 30 min after G{alpha}i1 was added to the assay tubes. The amount of monomeric G{alpha}i1 was proportional to the integrated area under the corresponding peak in the chromatogram. For the 0-min time point data, the peak area was assumed to represent 100% monomeric G{alpha}i1. The integrated peak area for the 30-min time point, normalized to the area under the corresponding 0-min time point, was taken to be the percentage of monomeric G{alpha}i1 remaining in solution.

Circular dichroism (CD) spectroscopy was used to determine the effects of hexanol on the secondary structure of G{alpha}i1. Spectra were collected on a temperature-controlled J-715 spectropolarimeter (JASCO, Tokyo, Japan) purged by nitrogen. Far-ultraviolet (185–240 nm) temperature-dependent measurements were performed with a 2-nm bandwidth by using a U-type quartz cell of path length 0.233 mm. CD spectra were recorded with five accumulations, each at a scan speed of 20 nm/min and a response time of 2 s. CD spectra were smoothed with a fast Fourier transform noise-reduction routine and are presented as molar ellipticity per residue ({theta}). Samples of G{alpha}i1 (~50 µM) in 10 mM potassium phosphate (pH 7.5), 100 mM NaCl, and 1 mM DTT with concentrations of hexanol varying from 0 to 30 mM were used in the measurements. The G{alpha}i1 concentration in the samples was determined spectrophotometrically by using a molar extinction coefficient of 44,670 ± 450 M-1cm-1 at 280 nm, which was determined with a nitrogen-digestion method (10). The CD spectrum was depicted as the difference between the spectrum of the protein in buffer and the spectrum of the buffer alone.

The {alpha}, ß, and unordered secondary structure contents of G{alpha}i1 were estimated from the CD spectra with SELCON3 (11), an algorithm for estimating the secondary structural content of proteins. SELCON is a self-consistent method that compares the CD spectrum of a protein with a set of reference protein spectra, for which the secondary structure content was determined from analysis of their crystal structure (11).

To determine the effect of hexanol on the heat denaturation of G{alpha}i1, the continuous temperature dependence of {theta} at 222 nm was measured by using a scan rate of 50°C/h and a response time of 8 s. At this wavelength, proteins in the native state have a much lower value for {theta} compared with their randomly structured denatured state. The temperature at which the protein denatures is determined from the midpoint temperature (Tmpt) of a discontinuity in {theta} as temperature increases. The Tmpt was determined as a variable from an NLS fitting of the data with the sigmoidal equation


In the equation, a and b are, respectively, the minimum and maximum observed {theta}, c is the slope of the change in {theta} at the Tmpt, and T is temperature.

For measurement of [35S]GTP{gamma}S binding to G{alpha}i subunits immunoprecipitated from ASM membranes, we reasoned that hexanol would not induce aggregation of G{alpha} in crude membrane preparations of ASM because the ability of hexanol to inhibit calcium sensitivity in intact ASM is reversible (6). This implies that the cellular environment has the necessary components to exhibit hexanol inhibition of G protein-coupled receptor (GPCR)-mediated calcium sensitivity, yet still prevent aggregation of G{alpha} due to hexanol. We assumed that many of the stabilizing influences would be present in the membrane where the G-protein heterotrimer is anchored. Thus, we examined the effect of hexanol on the intrinsic rate of [35S]GTP{gamma}S binding to endogenous G{alpha}i in crude membrane preparations of ASM.

The rate of [35S]GTP{gamma}S binding to endogenous G{alpha}i subunits derived from ASM was measured as described previously (9) and is briefly summarized. Suspensions of porcine tracheal smooth muscle membrane were prepared in 50 mM Tris-HCl (pH 7.4), 4.8 mM MgCl2, 2 mM EDTA, 100 mM NaCl, and 1 µM GDP. The supernatant consisted of a colloidal suspension of small membrane particles containing the G{alpha}iß{gamma} heterotrimeric complex. The protein concentration of the supernatant was diluted to 2.5 mg/mL. Supernatant (55 µL) was incubated at 30°C with or without 30 mM hexanol for 10 min. The reactions were initiated by the addition of 6 nM [35S]GTP{gamma}S ({approx}2.7 mCi/fmol) to each assay tube. The reactions were quenched after 1, 5, 10, 30, and 60 min with 0.6 mL of ice-cold IP buffer containing 50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 150 mM NaCl, 0.5% of the nonionic detergent IGEPAL CA630, 100 µM GDP, 100 µM GTP, and 20 µg/mL aprotinin. The suspensions were incubated with 70 µL of protein A-agarose beads that had been precoated with rabbit anti-G{alpha}i common anti-rabbit immunoglobulin (1:200 dilution) directed against an epitope at the C-terminus of G{alpha}i (Calbiochem, San Diego, CA). The pelleted beads were washed with 5 mL of IP buffer, transferred onto filters (Millipore, Bedford, MA), and washed with 20 mL of IP buffer. The dried filter paper retaining the beads was placed in a scintillation vial containing 4 mL of liquid scintillant (Ultima Gold; Packard Bioscience, Boston, MA) and counted. The nonspecific binding of [35S]GTP{gamma}S to the beads in the absence of protein was used as the blank value and was determined simultaneously for each study. The data from each binding curve were NLS curve-fit with the equation y = max(1 - e-kt), where max is the maximum amount of G{alpha}i bound to [35S]GTP{gamma}S, k is the rate of binding, and t is the time.

Results are presented as mean ± SD; n refers to the number of pigs used for the crude membrane preparations or the number of assay replicates for recombinant protein. The rates of [35S]GTP{gamma}S binding to endogenous G{alpha}i were determined from the curve fits to the IP data and were compared by paired Student’s t-test for significance. P < 0.05 was considered significant for all comparisons. SigmaPlot and SigmaStat (Jandel Scientific, San Rafael, CA), respectively, were used to perform the curve fitting and statistics.

[{gamma}32P]GTP was purchased from New England Nuclear (Boston, MA), and [35S]GTP{gamma}S was purchased from Amersham Pharmacia (Piscataway, NJ). Anti-G{alpha}i antibody was purchased from Calbiochem. Halothane was purchased from Ayerst Laboratories, Inc. (Madison, NJ). All other reagents were purchased from Sigma (St. Louis, MO).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
With the variables from the concentration-response curve fit, the G{alpha}i1-specific GTPase activity (max) was 0.029 mole Pi · mole G{alpha}i1-1 · min-1 (in agreement with the results of Linder et al.) (12), which was inhibited by hexanol with an IC50 of 22.3 mM (Fig. 1). This is a concentration of hexanol more than that needed to cause relaxation in ASM (6). However, at larger protein concentrations, solutions of G{alpha}i1 with hexanol became visibly turbid, leading us to suspect that hexanol induced protein aggregation.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. The effect of hexanol on the apparent GTPase specific activity (mole Pi · mole G{alpha}i1-1 · min-1) of recombinant G{alpha}i1. Data are the mean of three measurements with the associated SD.

 
Incubation of G{alpha}i1 subunits with hexanol (up to 30 mM) for 30 min at 30°C affected the chromatograms obtained by SEC-HPLC (Fig. 2A). This finding is consistent with aggregation of the subunits caused by hexanol. The area of the chromatographic peak of G{alpha}i1 measured after 30 min of hexanol exposure and normalized to the initial peak area is plotted in Figure 2B. This analysis provided an approximation of the percentage of G{alpha}i1 remaining in monomeric form after a 30-min incubation, which decreased in a manner dependent on the hexanol concentration. This finding suggests that the apparent inhibition of the recombinant G{alpha}i1 GTPase activity by hexanol is due at least in part to aggregation of monomeric subunits produced by hexanol. If it is assumed that the aggregated protein loses normal GTP binding and GTPase activity, this would decrease the amount of active G{alpha}i1 in solution.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. A, Monomeric G{alpha}i1 peak from the size-exclusion high-performance liquid chromatogram after a 30-min incubation at 30°C in 3, 10, and 30 mM hexanol. B, The integrated area under the G{alpha}i1 peak after the 30-min incubation (At30) normalized to the initial area (At0) as a function of hexanol concentration. The percentage of monomeric G{alpha}i1 in 30 mM hexanol is the mean ± SD of three measurements. The percentage of monomeric G{alpha}i1 at the other hexanol concentrations was determined from a single measurement.

 
SELCON3 (11) analysis of the CD spectra of G{alpha}i1 at 10°C (Fig. 3) determined that the secondary structure content was 36.1% {alpha}, 35.2% ß, and 28.8% unordered. Hexanol up to 30 mM did not affect this secondary structure (Table 1).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. The circular dichroism spectra of G{alpha}i1 with 10, 20, and 30 mM hexanol measured at 10°C. Hexanol does not have an obvious effect on the secondary structure of the protein, as estimated by SELCON3 analysis (Table 1).

 

View this table:
[in this window]
[in a new window]
  		Table 1. The Effect of Hexanol on the Secondary Structure of the G{alpha}i1 Subunit
 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. The effect of hexanol on the heat denaturation of G{alpha}i1, determined with circular dichroism spectroscopy. The denaturation temperature was assigned as the midpoint temperature of the increase in molar ellipticity ({theta}) and decreased from 47.2°C to 42.5°C between the control sample and the sample containing 30 mM hexanol (Table 1).

 
The effect of hexanol on the heat denaturation of G{alpha}i1 was investigated by monitoring {theta} at 222 nm as a function of temperature (Fig. 4). As the hexanol concentration increased to 30 mM, the Tmpt for G{alpha}i1 decreased from a maximum of 47.2°C to 42.5°C (Table 1). Thus, hexanol decreases Tmpt so that it approaches the temperature used in the GTPase assay (30°C). This apparently increases the rate of G{alpha}i1 aggregation at the assay temperature and is consistent with the aggregation observed with the SEC-HPLC measurements.

Regarding [35S]GTP{gamma}S binding to G{alpha}i subunits immunoprecipitated from ASM membranes preparations, the endogenous G{alpha}i subunit, isolated by IP from crude preparations of ASM membrane, bound [35S]GTP{gamma}S in a time-dependent fashion (Fig. 5). This reached a maximum value within approximately 10 min. Hexanol (30 mM) did not significantly affect the [35S]GTP{gamma}S binding rate (Fig. 5; n = 3; P = 0.20), suggesting that it neither caused aggregation nor affected the ability of nucleotide to associate with G{alpha}i subunits in this membrane preparation.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. The effect of 30 mM hexanol on the [35S]GTP{gamma}S-binding time course of endogenous G{alpha}i immunoprecipitated from the airway smooth muscle tissue preparation. Data are calculated from the mean curve-fit variables determined from three experiments (performed in duplicate) with the associated SD. The dashed curves are interpolated from the mean curve-fit variables. GTP = guanosine triphosphatase.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The major finding of this study was that at concentrations up to 30 mM, hexanol does not affect the rate of [35S]GTP{gamma}S binding to endogenous G{alpha}i, yet it induces aggregation of recombinant G{alpha}i1 in aqueous solution. In several systems, receptor activation of G protein-mediated pathways is inhibited by anesthetics (5,13–15), although it is not presently clear whether the G protein, the receptor, or the entire GPCR complex is the anesthetic target. Ishizawa et al. (7) photoaffinity-labeled rhodopsin-transducin, a GPCR complex, with [14C]halothane and noted that it did not appreciably incorporate into any elements of the G protein, including the G{alpha} subunit, but rather associated with the receptor. However, Pentyala et al. (8) reported that volatile anesthetics inhibited [35S]GTP{gamma}S binding to G{alpha} subunits in aqueous solution. It is unclear which elements of the GPCR complex interact with anesthetics to inhibit receptor activation of G proteins in ASM.

In ASM, G protein-coupled pathways regulate the amount of force produced for a given intracellular calcium concentration. The best-characterized system involves muscarinic receptors coupled to both G{alpha}i and G{alpha}q subunits (5,16,17). Both volatile anesthetics and hexanol inhibit receptor activation of this system (4–6,18). Our prior work suggested that the heterotrimeric G proteins coupled to muscarinic receptors were a potential target. We speculated that a direct effect on G{alpha} subunits was responsible, consistent with the observations of Pentyala et al. (8). However, we have been unable to reproduce their results by using a similar methodology; we found that halothane did not affect the rate of [35S]GTP{gamma}S binding to G{alpha}i subunits (9). This was true either with recombinant G{alpha}i1 studied in aqueous solution or with endogenous G{alpha}i from ASM membranes studied by using the IP technique used in this study. On the basis of preliminary data suggesting that hexanol did affect the GTPase activity of G{alpha}i1, we pursued this study.

The GTPase activity of the recombinant G{alpha}i1 was similar to that previously reported, supporting the validity of our assay techniques (12). The initial finding that hexanol reduces this activity confirmed our preliminary data. However, subsequent investigation with HPLC and CD spectroscopy revealed this finding to be an artifact of hexanol-induced aggregation of G{alpha}i1 in aqueous solution. Exploration of the stability of G{alpha}i1 revealed that the secondary structure of the monomer was not altered by hexanol. The lack of appreciable changes in the secondary structure of the protein in the presence of hexanol does not imply that there was no interaction between the protein and anesthetic, because changes in the higher-order structure might not affect the CD spectrum of G{alpha}i1. For example, the CD spectrum of albumin is not altered when it binds to halothane (19). However, hydrogen exchange measurements indicate that halothane has a stabilizing effect on albumin (20) but destabilizes myoglobin (20,21). Hexanol had a destabilizing effect on G{alpha}i1, decreasing its Tmpt enough to induce aggregation at the assay temperature of 30°C. A change in the rate of hexanol-induced G{alpha}i1 denaturation at 30°C is not obvious in Figure 4, perhaps because of the rapid heating rate experienced by the sample (50°C/h). The conclusion is drawn from the combined observations with CD and SEC-HPLC data. Although the GTPase concentration-response curve suggests that the GTPase activity was not inhibited until the hexanol concentration exceeded 10 mM (Fig. 1), evidence of aggregation observed with HPLC was seen at hexanol concentrations as small as 3 mM. The discrepancy probably reflects the fact that in the GTPase activity assay protocol, hexanol and subunit were in contact for only the 20 minutes necessary for the assay, whereas the HPLC data were obtained after 30 minutes of incubation.

Nucleotide-free G{alpha}i is readily susceptible to irreversible damage that prohibits further nucleotide exchange or hydrolysis activity and results in the aggregation of the isolated protein (22,23). This is similar to the effect of hexanol on isolated G{alpha}i. This might suggest that hexanol inhibited the nucleotide hydrolysis activity of isolated G{alpha}i1 by promoting the nucleotide free conformation, as Pentyala et al. (8) postulated, thus resulting in aggregation of the protein. If this is the case, it is not an effect common to all anesthetics because halothane does not induce aggregation or inhibit the nucleotide exchange activity of G{alpha}i subunits (9).

The absence of any effect of hexanol on the intrinsic rate of [35S]GTP{gamma}S binding to G{alpha}i from crude ASM membrane preparations suggests that aggregation does not occur in this environment. In this preparation, the G{alpha}i is present as the heterotrimer but is functionally uncoupled from the receptor (9). G{alpha}i may derive increased stability from the membrane and associated Gß{gamma} subunits, which could partially shield it from contact with the alcohol. However, the subunit is still able to exchange nucleotides in this conformation, which indicates that this molecule remains partially accessible to solutes, including hexanol. From crystal structures it is known that disordered regions of the G{alpha}i monomer adopt specific conformations when associated with the Gß{gamma} in the heterotrimer (24). Such associations with other proteins might structurally reinforce the subunit against aggregation and denaturation in the presence of hexanol. The range of hexanol concentrations examined in this study exceeds those necessary to produce anesthesia (50% effective concentration of approximately 0.75 mM) (25) or relaxation of ASM (50% effective concentration of approximately 1.3 mM) (18). The lack of effect of 30 mM hexanol on the intrinsic rate of [35S]GTP{gamma}S binding to G{alpha}i from native membranes and its lack of effect in smaller concentrations on GTPase activity of recombinant G{alpha}i1 suggest that the effects on calcium sensitivity in situ do not result from a direct inhibition of nucleotide exchange on this subunit. This finding implies either that the observed ability of hexanol to inhibit GPCR responses in situ is not mediated by an interaction of hexanol with the G{alpha}i subunit or that such an interaction requires an environment not present in the membrane preparation or the aqueous solution of isolated subunit.

The authors concluded that hexanol inhibited the GTPase activity of isolated G{alpha}i1 but not the nucleotide exchange activity of G{alpha}i assayed in crude ASM membrane preparations. The inhibition of isolated subunit was attributed to experimental artifact arising from protein aggregation, which is characteristic of this subunit (22). These results are consistent with earlier findings that halothane does not inhibit the nucleotide exchange activity of G{alpha}i, whether assayed as the isolated subunit or in crude ASM membrane preparations (9).


   Acknowledgments
 
Supported in part by Grants HL-45532 and HL-54757 from the National Institutes of Health (Bethesda, MD) and grants from the Mayo Foundation (Rochester, MN).

The authors thank Alfred G. Gilman MD, PhD, Professor of Pharmacology at the University of Texas Southwestern Medical Center in Dallas, TX, for generously supplying the G{alpha}i1; Franklyn G. Prendergast, MD, PhD, for the use of his spectroscopic equipment; and Kathy Street and Darrell Loeffler for expert technical assistance.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Eckenhoff RG, Shuman H. Halothane binding to soluble proteins determined by photoaffinity labeling. Anesthesiology 1993; 79: 96–106.[Web of Science][Medline]
  2. Curry S, Lieb WR, Franks NP. Effects of general anesthetics on the bacterial luciferase enzyme from Vibrio harveyi: an anesthetic target site with differential sensitivity. Biochemistry 1990; 29: 4641–52.[Medline]
  3. Slater SJ, Kelly MB, Larkin JD, et al. Interaction of alcohols and anesthetics with protein kinase Calpha. J Biol Chem 1997; 272: 6167–73.[Abstract/Free Full Text]
  4. Bremerich DH, Hirasaki A, Jones KA, Warner DO. Halothane attenuation of calcium sensitivity in airway smooth muscle: mechanisms of action during muscarinic receptor stimulation. Anesthesiology 1997; 87: 94–101.[Web of Science][Medline]
  5. Kai T, Jones KA, Warner DO. Halothane attenuates calcium sensitization in airway smooth muscle by inhibiting G-proteins. Anesthesiology 1998; 89: 1543–52.[Web of Science][Medline]
  6. Yoshimura H, Jones KA, Perkins WJ, Warner DO. Dual effects of hexanol and halothane on the regulation of calcium sensitivity in airway smooth muscle. Anesthesiology 2003; 98: 871–80.[Medline]
  7. Ishizawa Y, Sharp R, Liebman PA, Eckenhoff RG. Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling. Biochemistry 2000; 39: 8497–502.[Medline]
  8. Pentyala SN, Sung K, Chowdhury A, Rebecchi MJ. Volatile anesthetics modulate the binding of guanine nucleotides to the alpha subunits of heterotrimeric GTP binding proteins. Eur J Pharmacol 1999; 384: 213–22.[Web of Science][Medline]
  9. Streiff JH, Warner DO, Jones KA, Perkins WJ. Effect of halothane on the guanosine 5' triphosphate binding activity of G-protein {alpha}i subunits. Anesthesiology 2003; 99: 105–11.[Medline]
  10. Jaenicke L. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal Biochem 1974; 61: 623–7.[Web of Science][Medline]
  11. Sreerama N, Venyaminov SY, Woody RW. Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Protein Sci 1999; 8: 370–80.[Web of Science][Medline]
  12. Linder ME, Ewald DA, Miller RJ, Gilman AG. Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli. J Biol Chem 1990; 265: 8243–51.[Abstract/Free Full Text]
  13. Narayanan TK, Confer RA, Dennison RL, et al. Halothane attenuation of muscarinic inhibition of adenylate cyclase in rat heart. Biochem Pharmacol 1988; 37: 1219–23.[Web of Science][Medline]
  14. Schmidt U, Schwinger RH, Bohm M. Interaction of halothane with inhibitory G-proteins in the human myocardium. Anesthesiology 1995; 83: 353–60.[Medline]
  15. Nietgen GW, Honemann CW, Durieux ME. Influence of anesthetics on endogenous and recombinantly expressed G protein-coupled receptors in the Xenopus oocyte. Toxicol Lett 1998; 100–101: 319–27.
  16. Croxton TL, Lande B, Hirshman CA. Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle. Am J Physiol 1998; 275: L748–55.[Medline]
  17. Hirshman CA, Lande B, Croxton TL. Role of M2 muscarinic receptors in airway smooth muscle contraction. Life Sci 1999; 64: 443–8.[Web of Science][Medline]
  18. Sakihara C, Jones KA, Lorenz RR, et al. Effects of primary alcohols on airway smooth muscle. Anesthesiology 2002; 96: 428–37.[Web of Science][Medline]
  19. Johansson JS, Eckenhoff RG, Dutton PL. Binding of halothane to serum albumin demonstrated using tryptophan fluorescence. Anesthesiology 1995; 83: 316–24.[Medline]
  20. Eckenhoff RG, Tanner JW. Differential halothane binding and effects on serum albumin and myoglobin. Biophys J 1998; 75: 477–83.[Medline]
  21. Eckenhoff RG, Pidikiti R, Reddy KS. Anesthetic stabilization of protein intermediates: myoglobin and halothane. Biochemistry 2001; 40: 10819–24.[Medline]
  22. Zelent B, Veklich Y, Murray J, et al. Rapid irreversible G protein alpha subunit misfolding due to intramolecular kinetic bottleneck that precedes Mg2+ "lock" after GTP/GDP exchange. Biochemistry 2001; 40: 9647–56.[Medline]
  23. Ferguson KM, Higashijima T, Smigel MD, Gilman AG. The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins. J Biol Chem 1986; 261: 7393–9.[Abstract/Free Full Text]
  24. Sprang SR. G protein mechanisms: insights from structural analysis. Annu Rev Biochem 1997; 66: 639–78.[Web of Science][Medline]
  25. Fang Z, Ionescu P, Chortkoff BS, et al. Anesthetic potencies of n-alkanols: results of additivity and solubility studies suggest a mechanism of action similar to that for conventional inhaled anesthetics. Anesth Analg 1997; 84: 1042–8.[Abstract]
Accepted for publication September 18, 2003.




This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. A. Johnston, K. Afshar, J. T. Snyder, G. G. Tall, P. Gonczy, D. P. Siderovski, and F. S. Willard
Structural Determinants Underlying the Temperature-sensitive Nature of a G{alpha} Mutant in Asymmetric Cell Division of Caenorhabditis elegans
J. Biol. Chem., August 1, 2008; 283(31): 21550 - 21558.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Streiff, J.
Right arrow Articles by Jones, K. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Streiff, J.
Right arrow Articles by Jones, K. A.
Related Collections
Right arrow Mechanisms
Right arrow Pharmacology

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2004 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press