Volatile anesthetics have been found to interfere with the functioning of several G protein-coupled receptors, effects that may be relevant to the mechanism of anesthetic action. Lysophosphatidate (1-acyl-2-sn-glycero-3-phosphate; LP) is the simplest natural phospholipid. It has pronounced biological effects and signals through a specific G protein-coupled receptor. Because of its lipophilicity, the LP receptor is a feasible site of anesthetic interaction. Therefore, the authors investigated the effects of halothane and isoflurane on LP signaling using Xenopus oocytes.
Mature oocytes were harvested from Xenopus frogs, isolated, and defolliculated manually. Lysophosphatidate receptors are endogenously present in these cells. Angiotensin receptors were expressed recombinantly to study anesthetic effects on intracellular signaling. Oocytes were studied individually with a two-electrode voltage clamp at room temperature. Integrated Ca(2+)-activated Cl- currents (ICl(Ca)) were used to evaluate the effects of anesthetics on changes in intracellular Ca2+ concentration in response to receptor agonists (10(-7) M LP or 10(-7) M angiotensin II) or intracellular inositoltrisphosphate (IP3) injection.
Halothane depressed LP signaling in a concentration-dependent manner, with half-maximal inhibition at 0.23 mM and virtually complete inhibition at 0.34 mM. Responses could be recovered after an anesthetic-free wash. Oocyte injection with heparin, an IP3 receptor antagonist, completely blocked LP and angiotensin signaling, indicating similar IP3- dependent pathways. However, ICl(Ca) induced by angiotensin receptor activation or intracellular IP3 injection were not inhibited by halothane. Isoflurane, at comparable concentrations, did not depress LP responses in oocytes significantly.
Lipid-mediator signaling can be affected profoundly by volatile anesthetics. At clinically relevant concentrations, halothane and isoflurane have different effects on LP signaling. The inhibitory effects of halothane on the LP signaling pathway occur before the IP3 receptor.
Volatile anesthetics interact with various cellular systems, although the relative importance of these interactions to clinical anesthesia is largely unknown. Much recent research has focused on anesthetic-protein interactions. Effects on N-methyl-D-aspartate [1–3]and gamma amino-butyric acid receptor [2,4,5]signaling seem to offer the most promise for explaining the mechanism of general anesthesia. However, interactions with other systems may also be important, either in modulatory roles or by inducing anesthetic side effects.
Although anesthetic interactions with ion channel function have been studied in some detail, effects on second messenger-linked receptors have been investigated to a limited extent only. Nonetheless, studies have shown pronounced inhibition of, for example, muscarinic acetylcholine [6–9]and serotonin [9–11]receptor functioning, making the interactions between anesthetics and G protein-coupled receptors a worthwhile field of investigation. Of specific relevance are receptors for lipid mediators, because a hydrophobic ligand-binding domain might be a likely site of anesthetic action. The field of lipid mediators has attracted much attention recently and includes important messengers such as the prostaglandins and platelet-activating factor.
Lysophosphatidate (1-acyl-2-hydroxy-sn-glycero-3-phosphate; LP) is a novel lipid mediator, [12,13]originally known only as a precursor in de novo lipid synthesis. [14]However, LP also has several pronounced biological effects, including the induction of smooth muscle contraction, [15–17]cellular proliferation, [18–20]and platelet aggregation. [21–23]Many of these effects may be due to LP's pronounced ability to induce actin stress fiber reorganization, and they are mediated through a specific G protein-coupled receptor for the compound. [24]Although some volatile anesthetics exhibit effects opposite to those of LP signaling in various cell types, suggesting an interaction, and although the lipophilicity of LP makes its receptor a feasible site for such interaction, the direct anesthetic effects on the LP signaling system have not been studied.
We hypothesized that LP signaling can be inhibited by volatile anesthetics. To test this hypothesis, we studied the effects of halothane and isoflurane on LP signaling in Xenopus laevis oocytes. Xenopus oocytes form a good model to study the compound because these cells express a well-described endogenous LP signaling system [25–28]coupled to intracellular Ca2+ release (Figure 1(A)). In addition, the presence of Ca2+-activated Cl sup - channels allows convenient measurement of this Ca2+ release by electrophysiologic means. [26,29]In this study, we tried to answer the following questions:
1. Do volatile anesthetics, at clinically relevant concentrations, affect LP signaling in oocytes?
2. If so, are these differences in effect between halothane and isoflurane, two anesthetics used frequently in clinical practice?
3. Can these effects be localized to a specific of the signaling pathway?
Materials and Methods
Animals
The study protocol was approved by the Animal Research Committee at the University of Virginia. Female Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI), housed in an established frog colony, and fed regular frog brittle twice per week. To remove the oocytes, each frog was anesthetized by immersion in 0.2% methanesulfonate salt solution until unresponsive to a painful stimulus (toe pinching). A 1-cm-long incision was made in a lower abdominal quadrant and a lobule of ovarian tissue, containing approximately 200 oocytes, was removed and placed in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO sub 3, 0.41 mM CaCl2, 0.82 mM MgSO4, 0.3 mM Ca2NO3, 15 mM HEPES, and 0.1 mg/ml gentamicin, pH adjusted to 7.4). The wound was closed in two layers and the frog was allowed to recover from anesthesia, kept in a separate tank overnight, and returned to the colony the next day.
The ovarian tissue was immediately and copiously washed in modified Barth's solution and dissected into small clusters of 20 to 50 oocytes. Mature oocytes (Dumont stages V and VI) were isolated, defolliculated manually with microforceps, and maintained in modified Barth's solution.
Materials
Lysophosphatidate (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) stock was obtained from Avanti Polar Lipids (Alabaster, AL) and diluted in a 1% solution of fatty acid-free bovine serum albumin (BSA; ICN Pharmaceuticals, Costa Mesa, CA) to appropriate concentrations. Halothane and isoflurane were obtained from Halocarbon Laboratories (River Edge, NJ) and Ohmeda PPD (Liberty Comer, NJ), respectively. All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
Electrophysiologic Recording
A single defolliculated oocyte was placed in a perfusable recording chamber (3-ml volume) filled with Tyrode's solution (150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.2 mM CaCl2, 10 mM dextrose, 10 mM HEPES, pH adjusted to 7.4). Microelectrodes were pulled in one stage from capillary glass (BBL with fiber; World Precision Instruments, Sarasota, FL) on a micropipette puller (model 700C; David Kopf Instruments, Tujunga, CA). Electrode tips were broken to a diameter of approximately 10 micro meter, providing a resistance of 1 to 3 M Omega, and filled with 3 M KCl. The cell was voltage clamped using a two-microelectrode oocyte voltage-clamp amplifier (OC725A; Warner Corp., New Haven, CT) connected to a data acquisition and analysis system running on an IBM-compatible personal computer. The acquisition system consisted of a DAS-8 A/D conversion board (Keithley-Metrabyte, Taunton, MA), and analysis was performed with OoClamp software. [30]Holding potential was -70 mV, and only cells exhibiting stable holding currents less than 1 micro Ampere during a 1-min equilibration period were included in analysis. Membrane current was sampled at 125 Hz and recorded for 5 s before and 55 s after application of the appropriate receptor agonist. Agonists were delivered in 30-micro liter aliquots for a period of 1 to 2 s using a hand-held micropipettor positioned approximately 5 mm from the oocyte. Unless otherwise indicated, washout did not occur during the time scale of traces presented. To prevent desensitization of the receptor or signaling pathways from influencing the results, each oocyte received only a single agonist application. Responses were quantified by integrating the current trace by quadrature and are reported in microcoulombs (micro C). All experiments were performed at room temperature.
Anesthetic Administration
To determine the effects of halothane and isoflurane on LP-induced currents, output from the appropriate vaporizer was bubbled through a reservoir filled with 30 ml Tyrode's solution and allowed to equilibrate for at least 10 min. Air at a flow rate of 500 ml/min was used as the carrier gas. After equilibration, the solution from the reservoir was perfused through the recording chamber at a rate of approximately 10 ml/min, and measurements were obtained after ten bath volumes had been exchanged (3 min). The concentrations of the anesthetics in the recording chamber were quantified by gas chromatography: Triplicate samples from the recording chamber were equilibrated with air and analyzed in a gas chromatograph (Aerograph 940; Varian Analytical Instruments, Walnut Creek, CA) calibrated with the appropriate standard. Results were converted to concentrations in liquid using Kreb's solution/air partition coefficients at 25 degrees Celsius [31]and to corresponding partial pressure (vol%) at that temperature. [32]Each oocyte was exposed to a single concentration of anesthetic only.
Intracellular Injection
To study inositoltrisphosphate (IP3)-induced Ca2+-activated Cl sup - currents (ICl(Ca)), a third microelectrode, connected to an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA), was inserted into the oocyte under voltage clamp. Oocytes were injected with 50 nl IP3(final concentration, 100 micro Meter) and adequacy of injection was confirmed by noting a small increase in cell size. Membrane currents were recorded for 5 s before and 55 s after IP3injection. Microinjection pipettes were examined for obstruction before and after recording. Only experiments in which tips were fully functional before and after injection were included in the analysis.
For intracellular injection of heparin, an automated microinjector (Nanoject), was used. Oocytes were injected with 60 nl heparin (final concentration, 120 ng/micro liter) at least 30 min before agonist application. Adequacy of injection was confirmed by observing the small increase in cell size on injection.
mRNA Synthesis and Injection
The rat AT1Aangiotensin II receptor clone was obtained from Dr. K. R. Lynch (University of Virginia, Charlottesville, VA) as a cDNA of 1.2 kilobasepair in the CDM8 vector (Invitrogen, San Diego, CA). The construct was linearized with the nuclease Xho I and transcribed in the presence of a capping analog by T7 polymerase.
Oocytes were injected with 50 nl mRNA (3–5 ng) in water using an automated microinjector (Nanoject). The adequacy of injection was confirmed by noting the slight increase in cell size during injection. The cells were cultured in modified Barth's solution for 3 days before study.
Data Analysis
Responses are reported as means +/- SEM. Differences among treatment groups were analyzed using the Student's t test. If multiple comparisons were made, data were analyzed using analysis of variance followed by a t test corrected for multiple comparisons. Probability values less than 0.05 were considered significant. Concentration-response curves were fit to the following logistic function, derived from the Hill equation:Equation 1where Ymaxand Yminare the maximum and minimum obtained response, n is the Hill coefficient, and X50is the concentration at which the half-maximal response occurs.
Results
Lysophosphatidate Induces an Inward Current in Oocytes
Whereas application of 1% BSA had no effect on oocytes at a holding potential of -70 mV (Figure 1(B)), application of 10 sup -7 M LP (approximately the median effective concentration in this model [33]), diluted in 1% BSA, resulted in transient inward currents of several microamperes. The current developed after a short latency period (less or equal to 5 s) and consisted of a fast inward component followed by a relaxation over several seconds with superimposed small fluctuations. We and others have shown previously that the oocyte responses to LP are due to release of Ca2from intracellular stores and subsequent activation of ICl(Ca). [25,26,28]The shape of the response is indistinguishable from responses induced by activation of other G protein-coupled receptors.
Halothane Reversibly Inhibits Lysophosphatidate Responses in Oocytes
We tested the ability of halothane to interfere with LP signaling. Halothane at clinically relevant concentrations depressed LP-induced ICl(Ca) in a concentration-dependent manner (Figure 2(A and B). In the absence of halothane, average charge movement induced by 10 sup -7 M LP was 1.6 +/- 0.2 micro C. In the presence of 0.34 mM (0.64%) halothane, responses to LP were inhibited completely. Curve fitting using the Hill equation revealed a half-maximal inhibitory concentration of 0.23 +/- 0.02 mM (0.43%) and a Hill coefficient of 6.3 +/- 2.5.
To determine if the effect of halothane was reversible, LP-induced currents were tested under three conditions: in the absence and presence of halothane and after exposure to halothane followed by a wash with anesthetic-free solution. Three separate treatment groups were used to prevent multiple agonist applications from desensitizing the LP receptor or intracellular signaling pathways. A high concentration of halothane was used because it was considered more likely to cause irreversible effects. Although LP-induced responses were depressed more than 95% in the presence of anesthetic, ICl(Ca) obtained in response to LP after exposure to 0.34 mM (0.64%) halothane and a wash with anesthetic-free solution were similar in size to control responses (Figure 2(C)).
Halothane Does Not Inhibit Angiotensin Responses Oocytes
In a previous study, we showed that 0.34 mM (0.64%) halothane does not affect responses to 10 sup -7 M angiotensin II (ATII) in oocytes expressing the AT1Aangiotensin receptor. [6]Apart from the receptor and possibly the G protein, LP and AT1Areceptors are presumed to signal through the same intracellular pathway, because they both induce Ca2+ release and Ca2+-activated Cl sup - channel activation and because the oocyte contains only a single molecular species of phospholipase C coupling receptor activation to intracellular Ca2+ release. [34]Therefore, as halothane had no inhibitory effect on angiotensin signaling, halothane's site of action is unlikely to be downstream of the LP receptor and G protein. To confirm our previous data, we expressed AT1Areceptors in oocytes and tested the ability of 0.34 mM (0.64%) halothane to inhibit ICl(Ca) induced by 10 sup -7 M ATII (as for LP, this concentration is approximately half-maximal in this model [35]). As in our previous investigation, halothane at this concentration had no effect (Figure 3). We proceeded to confirm that LP and AT receptors signal through the same transduction pathway and that halothane has no effect on this common signaling pathway.
Lysophosphatidate and Angiotensin Receptors Signal through Inositoltrisphosphate
Both LP [36,37]and angiotensin signaling [38]result in IP3release. To verify that these receptors indeed signal through this mechanism in our model, we injected heparin (MW = 3,000; 60 nl; final concentration, 120 ng/micro liter) into oocytes and tested their ability to respond to agonist application 30 min later. Heparin antagonizes the effect of IP3on its receptor and inhibits IP3-mediated Ca2+ responses in oocytes. [38]To ensure that the intracellular injection process did not impair the signaling ability of the cells, some oocytes were injected with 60 nl water, which was found to have no effect on LP or ATII-induced ICl(Ca)(Figure 4(A)). However, responses to LP and ATII were blocked significantly by previous heparin injection (Figure 4(B and D)). In contrast, the de-N-sulfated form of heparin, which is a less potent IP3antagonist, [38]did not affect agonist-induced responses significantly (Figure 4(C)).
Halothane Does Not Affect Inositoltrisphosphate Responses in Oocytes
Because lack of effect on AT signaling suggests that halothane does not affect the signaling pathway downstream of receptor or G protein, we wanted to confirm that the anesthetic does not affect the intracellular Ca2+ release system or the Ca2+-activated Cl sup - channel. For this purpose we injected IP3into oocytes and determined the effect of halothane on ICl(Ca) induced by the compound. Inositoltrisphosphate (50 nl; final concentration, 100 micro Meter) induced currents similar in kinetics to those observed with LP or AT1Asignaling (Figure 5(A)). In preliminary experiments, we established that this concentration of injected IP3resulted in response sizes similar to those observed with 10 sup -7 M LP or ATII. To ensure that the observed IP3-induced currents were not a result of the injection process, membrane currents were recorded after injections of 50 nl water. Intracellular injection of water did not induce any currents (first trace;Figure 5(A)). The average IP3-induced response was 4.2 +/- 0.5 micro C (n = 22). Halothane (0.34 mM; 0.64%) had no effect on these responses (Figure 5(A and B); average charge movement, 4.2. +/- 0.6 micro C; n = 20), indicating that the pathway downstream of IP3generation is unaffected by the anesthetic.
Isoflurane Does Not Depress Lysophosphatidate Responses in Oocytes
We also investigated the effects of isoflurane on LP-induced Cl sup - currents. In contrast to halothane, isoflurane caused only modest depression of LP-induced currents in oocytes (Figure 6). The calculated half-maximal concentration was 0.22 +/- 0.07 mM (0.46%), with a Hill coefficient of 3.4 +/- 2.9. Maximal inhibition was 45%, which was not statistically different from control (2.7 +/- 0.3 micro C). Thus the effects of these two anesthetics on the LP response in oocytes are quite different.
Discussion
In this study we show that halothane at clinically relevant concentrations completely inhibits LP, but not AT, responses in Xenopus oocytes. In contrast, equivalent concentrations of isoflurane induced no significant inhibition of LP signaling.
Effects and Site of Action of Lysophosphatidate
Lysophosphatidate, the simplest natural phospholipid, has many biological effects. Examples include contraction of vascular [39]and other [15]smooth muscle; aggregation of human and feline platelets [21]; rapid neurite retraction in several neuronal cell lines [28,40,41]*; mitogenesis in fibroblasts [18,19]through a mechanism involving p21rasand the mitogen-activated protein (MAP) kinase pathway [41,42]; and inhibition of proliferation in myeloma cells. [43]Many of these diverse effects presumably can be reduced to a primary action on the intracellular actin stress fiber system through an effect on the guanine triphosphate (GTP)-binding protein Rho. [24,44]In addition, the compound inhibits cyclic adenosine monophosphate generation and induces Ca2+ release from intracellular stores through IP3generation. In Xenopus laevis oocytes, we and other investigators found that LP induces ICl(Ca) through this mechanism. [25–28]The compound is released into serum by activated platelets, [45,46]and its importance is demonstrated by the fact that many of the studies we refer to have shown that well-known serum effects such as neurite retraction and actin reorganization are, in fact, due to LP bound to serum albumin.
Although it has not been cloned yet, many studies document the existence of an LP receptor that couples to a G protein signaling cascade. When microinjected into cells, LP has no effect, implying an extracellular site of action. [25,26,36,47]Additional evidence for a membrane receptor comes from work done by van der Bend et al., [48]who, using a photoreactive, radiolabeled, cross-linking LP analog, identified a putative LP membrane receptor of 38–40 kd. In addition, responses to LP are blocked by suramin, an inhibitor of many ligand-receptor interactions. Several studies support a G protein-coupled signaling system for LP. Transient increases in Ca2+ levels in fibroblasts [18,36]and oocytes [25,26]induced by LP have kinetics typical of those of G protein-coupled receptor responses. In addition, some LP-induced responses are sensitive to pertussis toxin, [18,26]and GTP gamma S, a nonhydrolyzable GTP analog, potentiates the effects of LP. [18,37]Lysophosphatidate action is highly specific. Virtually no structurally related compounds induce changes in Ca2+ levels in human fibroblasts or oocytes. Prostaglandins and platelet-activating factor have been shown clearly to signal through other receptor classes. [49,50]
Site of Anesthetic Inhibition
The inhibition of LP responses by halothane observed in the present study is likely to occur at the receptor or G protein level. Suggestive evidence for this derives from our finding that angiotensin signaling is not inhibited by halothane concentrations that virtually abolish LP responses. The two agonists are presumed to signal through the same intracellular pathway and have similar potencies in our model. A single phospholipase C type has been shown to be involved in receptor-mediated Ca2+ signaling in oocytes, [34]making Ca2+ release from similar pools and activation of the same CI sup - channel very likely. The indistinguishable response kinetics in these cells supports this. Our heparin microinjection experiments show that both LP and AT1Areceptors signal through IP3-sensitive Ca2+ stores. Furthermore, no other intracellular calcium release channels, such as ryanodine receptors, have been found in oocytes. [38]Therefore, apart from the receptors, and possibly the coupled G proteins, AT and LP signaling pathways can be considered identical, and lack of halothane on one, but not the other, pathway suggests an effect at the receptor level.
Additional evidence supporting lack of anesthetic interference with intracellular signaling mechanisms derives from our finding that IP sub 3 -induced currents are not affected by halothane. This indicates that the anesthetic does not affect the Ca2+ release mechanism or the Ca sup 2+-activated Cl sup - channel. Our findings correspond with other studies in which low concentrations of halothane were shown not to affect IP3-linked Ca2+ signaling. For example, Smart et al. [51]studied the effect of halothane (0.5–1%) on basal and carbachol-stimulated IP3production in SH-SY5Y human neuroblastoma cells and found no inhibition but rather a modest enhancement of IP3formation in the presence of anesthetic. Hossain and Evers [52]and Stern et al. [53]observed that halothane (0.5 mM) had almost no effect on thyrotropin-releasing hormone-induced, IP3-mediated intracellular Ca sup 2+ release.
Our data cannot rule out an effect of halothane on the LP molecule itself or on LP binding to albumin. However, such interactions are unlikely to explain our results, because volatile anesthetics also inhibit signaling through G protein-coupled receptors with agonists that are less hydrophobic and do not require albumin for solubilization. We have shown that halothane significantly inhibits signaling through muscarinic acetylcholine receptors. [6]Lin et al. [9]reported findings similar to ours, showing that although enflurane (1.8 mM) inhibited muscarinic and serotonin signaling, it did not affect the signaling cascade downstream of phospholipase C. A differential effect of isoflurane and halothane on LP binding to albumin is also unlikely. Although volatile anesthetics have been shown to compete with fatty acids for binding sites on albumin, halothane and isoflurane have similar binding constants: Kdwas 1.4 mM for isoflurane and 1.3 mM for halothane. [54,55]Therefore, our data are most compatible with an effect of halothane on the LP receptor or its associated G protein.
Anesthetic Effects Suggestive of Lysophosphatidate Inhibition
Besides being a useful model for investigating anesthetic-protein interactions, LP is an important compound to study because it is a likely regulator of cell proliferation, particularly in tumor cells. The lack of selective LP antagonists limits our understanding of this growth modulator, although recent reports, [33,56]if confirmed, suggest that this problem may be resolved. Suramin inhibits LP responses, but it is nonspecific because it blocks many other growth factor actions. Many studies have shown volatile anesthetics to exhibit effects opposite of those of LP signaling, although some reports are conflicting. As noted earlier, LP is a mitogen and induces stress fiber assembly. Halothane inhibits growth of neuroblastoma cells in a dose-dependent manner by disrupting actin-like microfilaments of the mitotic apparatus. [57]Similar results were found in other cell types. Halothane also affects the distribution pattern of actin in fibroblasts and neuroblastoma cells [58]and prevents cell cleavage in sea urchin eggs, [59]another actin-dependent process. Both enflurane and halothane at equivalent minimum alveolar concentrations interfere with cell division in Tetrahymena pyriformis. [60]As previously noted, LP is a potent platelet aggregator and decreases cyclic adenosine monophosphate levels in many cell types. In contrast, halothane has been found to decrease aggregation in human platelets [61,62]accompanied by increased cyclic adenosine monophosphate levels. [61]Thus the LP antagonist-like properties and some side effects of volatile anesthetics may be due to interference with LP signaling.
Conclusions
Our findings show that lipid mediator signaling can be affected profoundly by anesthetics. It should be determined if prostaglandins and platelet-activating factor are similarly inhibited by volatile anesthetics. It is not possible to study direct interactions between anesthetics and the LP receptor, because a binding assay for the LP receptor has not been established. If, however, anesthetic interference with signaling occurs at the LP binding site, the simple molecular structure of many anesthetics might allow elucidation of the molecular determinants that define an LP antagonist. Finally, because the LP receptor is G protein linked, our findings provide additional support for anesthetic interference with G protein-coupled receptors.
*Jalink K, Moolenaar WH: Lysophosphatidate and thrombin induce membrane depolarization in NIE-115 neuroblastoma cells; lysophosphatidate as an extracellular messenger; a study of early cellular effects and mechanism of action. Doctoral thesis, Jalink K. Leiden, the Netherlands, 1993, pp 81–7.