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Distinct molecular targets for the central respiratory and cardiac actions of the general anesthetics etomidate and propofol

Anja Zeller^*, Margarete Arras, Anelise Lazaris^*, Rachel Jurd^* and Uwe Rudolph^*,1

^* Institute of Pharmacology and Toxicology and
Institute of Laboratory Animal Science, University of Zürich, Switzerland

¹ Correspondence: Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstr. 190, CH-8057 Switzerland. E-mail: rudolph{at}pharma.unizh.ch

SPECIFIC AIMS

The targets mediating specific actions of general anesthetics can be identified by introducing mutations into the mouse germline rendering a specific neurotransmitter receptor subtype insensitive to selected agents. Studying ß3(N265M) mice has revealed that the immobilizing action and, in part, the hypnotic action of etomidate and propofol are mediated by ß3-containing GABA_A receptors. The pharmacological spectrum of these general anesthetics also includes respiratory depression, cardiac depression and hypothermia, but it is unknown by which targets these actions are mediated. To investigate the potential involvement of ß3-containing GABA_A receptors in these actions, we compared the effects of etomidate and propofol in ß3(N265M) and wild-type mice.

PRINCIPAL FINDINGS

1. Intravenous anesthetics-induced respiratory depression is absent in ß3(N265M) mice
To identify targets mediating the respiratory depressant action of the general anesthetics etomidate and propofol, arterial blood gases and pH values were determined after intravenous injection of these agents in ß3(N265M) and wild-type mice (Fig. 1 ). After i.v. injection of 15 mg/kg etomidate or 40 mg/kg propofol the oxygen partial pressure (paO₂) is dramatically decreased in wild-type mice, compared with ß3(N265M) mice. Likewise, in wild-type mice the carbon dioxide partial pressure (paCO₂) is substantially increased by these agents compared with ß3(N265M) mice. In contrast, the actions of alphaxalone on paO₂ and paCO₂ are indistinguishable between genotypes, demonstrating that the ß3(N265M) mice respond normally to general anesthetics whose action is not affected by the N265M point mutation in vitro.

View larger version (20K): [in this window] [in a new window]   Figure 1. Assessment of anesthetic-induced respiratory depression by blood gas analysis. A, B) In ß3(N265M) mice injected with etomidate and propofol, paO2 was higher and paCO2 was lower compared with wild-type mice, indicating the dependence of the respiratory depressant effects of these anesthetics on ß3-containing GABAA receptors. The neurosteroidal anesthetic alphaxalone (used in a mixed preparation with alphadolone, Saffan®), whose action is not affected by the ß3(N265M) mutation in vitro, elicits changes in blood gases without a difference between genotypes. C) Similarly, after etomidate and propofol, but not after alphaxalone, pH was higher in ß3(N265M) mice compared with wild-type. The horizontal green lines indicate normal values. n = 10. *P < 0.05, **P < 0.01, ***P < 0.001.

Wild-type mice develop a much stronger acidosis than ß3(N265M) mice after administration of etomidate and propofol, whereas the pH after injection of alphaxalone was indistinguishable between genotypes. Thus, both blood gas analysis and pH measurement revealed that the respiratory depressant effects of etomidate and propofol are mediated by ß3-containing GABA_A receptors.

2. Intravenous anesthetics decrease heart rate in ß3(N265M) and wild-type mice
To evaluate whether the cardiac depressant effect of etomidate and propofol is mediated by ß3-containing GABA_A receptors, heart rate was monitored using a radiotelemetry system in unrestrained animals. Etomidate and propofol decreased the heart rate significantly in both genotypes (Fig. 2 ). After i.v. injection of 10 mg/kg etomidate the decrease in heart rate is marginally larger in wild-type than in ß3(N265M) mice, and after i.p. injection of etomidate there is a significantly larger decrease in heart rate in wild-type vs. ß3(N265M) mice. After i.v. injection of alphaxalone and propofol, and also after i.p. injection of propofol, the decrease in heart rate is similar in wild-type and ß3(N265M) mice.

View larger version (43K): [in this window] [in a new window]   Figure 2. Anesthetic-induced heart rate depression. With injection of general anesthetics heart rate (HR) decreases in both wild-type and ß3(N265M) mice. A) Maximum HR change after injection of anesthetic or vehicle compared with 1 h baseline before injection. B) Time course of HR change after injection of etomidate i.v.. Group sizes: propofol i.v.: wt n = 7, ß3(N265M) n = 5, etomidate i.v.: wt n = 7, ß3(N265M) n = 7, alphaxalone(Saffan®) i.v.: wt n = 6, ß3(N265M) n = 6, propofol i.p.: wt n = 4, ß3(N265M) n = 6, etomidate i.p.: wt n = 5, ß3(N265M) n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Etomidate and propofol depress the heart rate in both ß3(N265M) and wild-type mice, indicating that targets other than ß3-containing GABA_A receptors primarily mediate this effect, and the genotype difference for etomidate suggests that ß3-containing GABA_A receptors play a minor role in mediating its heart rate depressant effects.

3. Intravenous anesthetics induce hypothermia in ß3(N265M) and wild-type mice
General anesthetics are known to cause hypothermia. To evaluate the role of ß3-containing GABA_A receptors in hypothermia, we monitored core body temperature (CBT) changes in response to etomidate and propofol in ß3(N265M) mice and wild-type mice telemetrically. After injection of all anesthetics tested the CBT decreased significantly in both genotypes compared with the baseline before injection. In the drug-injected mice the temperature decreases steadily to a minimum and starts to increase at about the same time that mice regain the righting reflex (data not shown).

After injection of 10 mg/kg etomidate i.v., CBT decreased significantly less in ß3(N265M) compared with wild-type mice (wt: 4.9°C, ß3(N265M) 3.4°C, P=0.010), whereas after injection of 20 mg/kg etomidate i.p. hypothermia is marginally less pronounced in ß3(N265M) mice compared with wild-type mice (wt: 6.3°C, ß3(N265M) 5.1°C; P=0.058). After injection of 30 mg/kg propofol i.v. (wt 2.6°C, ß3(N265M) 2.5°C, P=0.952) and after injection of 180 mg/kg propofol i.p. (wt: 8.9°C, ß3(N265M) 6.2°C; P=0.071) the decrease in body temperature is similar in wild-type and ß3(N265M) mice.

As observed for respiratory and cardiac depression, the hypothermic action of alphaxalone is similar in both genotypes (wt: 3.1°C, ß3(N265M) 2.6°C; P=0.296)

Etomidate and propofol induce hypothermia in both ß3(N265M) and wild-type mice, and the difference between genotypes for etomidate indicates only a minor role for ß3-containing GABA_A receptors in mediating etomidate-induced hypothermia.

4. The sedative action of etomidate is independent of ß3-containing GABA_A receptors
The sedative action of general anesthetics at subanesthetic doses may contribute to hangover effects during the recovery phase. In this study, we investigated whether the sedative action of etomidate is also mediated by ß3-containing GABA_A receptors.

Both genotypes display a decrease in motor activity after administration of 5 mg/kg (WT: P<0.05, ß3(N265M): P<0.01) and 10 mg/kg (WT: P<0.01, ß3(N265M) <0.001) etomidate compared with vehicle. Furthermore, there is no significant difference between genotypes for either vehicle or any dose of etomidate. Thus, the sedative action of etomidate is present in ß3(N265M) mice, indicating that ß3-containing GABA_A receptors do not mediate this effect.

CONCLUSIONS AND SIGNIFICANCE

In this report, we investigated the contribution of ß3-containing GABA_A receptors to various physiological and behavioral end points of the general anesthetics propofol and etomidate. We show that the respiratory depressant effect of propofol and etomidate is mediated by ß3-containing GABA_A receptors whereas the cardiac depressant effect, hypothermia, and sedation are largely not mediated by ß3-containing GABA_A receptors.

The general anesthetics propofol and etomidate strongly reduce respiration in wild-type mice, whereas there is almost no such effect in ß3(N265M) mice. The oxygen partial pressure in wild-type mice is reduced to almost 50% compared with ß3(N265M) mice. The carbon dioxide partial pressure is increased in wild-type mice twice as much as in ß3(N265M) mice. We used the same doses of propofol and etomidate that in our previous study were lethal for 50% of wild-type mice, but for none of the ß3(N265M) mice. In contrast, after injection of 30 mg/kg alphaxalone the oxygen partial pressure is similarly reduced in both wild-type and ß3(N265M) mice, and the carbon dioxide partial pressure is similarly increased. This dose of alphaxalone was lethal for 50% of both wild-type and ß3(N265M) mice in our previous study. The anesthetic-induced heart rate depression is quite substantial in both genotypes (e.g., 44% after i.v. injection of etomidate), and our results suggest that the cause of anesthetic-induced death in wild-type animals is respiratory depression and not cardiac depression, since respiratory depression but not cardiac depression is absent in ß3(N265M) mice.

After injection of etomidate, hypothermia is less pronounced in ß3(N265M) mice than in wild-type mice; for propofol, the hypothermic response was indistinguishable between ß3(N265M) mice and wild-type mice, consistent with recent findings by others that the hypothermic response is mediated largely by ß2-containing GABA_A receptors.

For all parameters analyzed so far (i.e., immobility, hypnosis, hypothermia, cardiac depression, and respiratory depression) the action of alphaxalone was indistinguishable in ß3(N265M) mice and wild-type mice, indicating that, as predicted from in vitro studies, ß3(N265M)-containing GABA_A receptors are sensitive to neurosteroidal anesthetics.

At subanesthetic doses, as they occur during recovery from anesthesia, general anesthetics have a sedative effect. Here we describe that etomidate decreases motor activity in both ß3(N265M) mice and wild-type mice, indicating that this action is mediated by targets distinct from ß3-containing GABA_A receptors, in line with an earlier report by others that subanesthetic sedative actions of etomidate are mediated by ß2-containing GABA_A receptors.

In summary, all data available for etomidate point to ß3-containing GABA_A receptors mediating immobility and respiratory depression, in part hypnosis and to a minor degree hypothermia, while ß2-containing GABA_A receptors mediate hypothermia (for the most part), in part hypnosis and sedation. Based on our results, we hypothesize that the cardiac depressant actions of etomidate may be mediated by ß2-containing GABA_A receptors. For propofol, we know that ß3-containing GABA_A receptors mediate immobility, respiratory depression, and in part hypnosis whereas distinct targets, most likely ß2-containing GABA_A receptors, mediate hypnosis (in part) and hypothermia.

With respect to future drug development, our data indicate that anesthetic agents with a high degree of selectivity for ß3-containing GABA_A receptors over ß2-containing GABA_A receptors provide immobility and respiratory depression (which easily can be taken care of clinically) but would produce significantly less cardiovascular depression, hypothermia, and sedation during the recovery phase. Our data demonstrate that immobilizing and cardiac depressant actions of intravenous general anesthetics are indeed mediated by different targets and thus can be separated pharmacologically, enabling the development of general anesthetic agents with an improved therapeutic profile.

View larger version (18K): [in this window] [in a new window]   Figure 3. Proposed roles of etomidate and propofol on GABAA receptor subtypes. These assignments are based on the following tests: immobility: lost of hind limb withdrawal reflex; respiratory depression: increase in paCO2 and decrease in paO2 and pH; hypnosis: loss of righting reflex; sedation: decrease in motor activity; hypothermia: decrease in core body temperature; cardiac depression: decrease in heart rate. Data are based on this study and previous work.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3443fje; doi: 10.1096/fj.04-3443fje

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