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Anesth Analg 1999;89:225
© 1999 International Anesthesia Research Society

GENERAL ARTICLES

Volatile Anesthetics Reduce Calcium Current in Parasympathetic Neurons from Bullfrog Hearts

Koki Hirota, MD, Akira Masuda, MD, and Yusuke Ito, MD

Department of Anesthesiology, Toyama Medical and Pharmaceutical University School of Medicine, Toyama, Japan

Address correspondence and reprint requests to Dr. Hirota, Department of Anesthesiology, Toyama Medical and Pharmaceutical University School of Medicine, 2630 Sugitani, Toyama 930-0194, Japan.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Although the autonomic nervous system regulates cardiac function, the cellular mechanism(s) of general anesthetics on the activities of parasympathetic neurons have not been directly assessed. We therefore studied the volatile anesthetic actions on the Ca2+ current of parasympathetic neurons isolated from bullfrog hearts. Neurons were enzymatically isolated from the interatrial septum of bullfrog heart and maintained in a short-term tissue culture. The Ca2+ current was recorded with a whole-cell voltage-clamp method under a Na+, K+-free condition. Isoflurane (2.5 vol%) and sevoflurane (5.0 vol%) reduced the peak amplitude of the Ca2+ current (to 79% and 72% of control, respectively) without changing the reversal potential. The curve-fit analysis of the inactivation kinetics revealed that isoflurane and sevoflurane accelerated the inactivation of the current and that isoflurane shifted the midpoint of the steady-state inactivation curve of the Ca2+ current toward negative by 13.6 mV. The results indicate that volatile anesthetics reduce the Ca2+ current of parasympathetic neurons and modify the inactivation kinetics.

Implications: The anesthetic reduction of the Ca2+ current of parasympathetic neurons can induce a decrease of acetylcholine release from the postganglionic endings. These findings, in part, account for the anesthetic attenuation of the vagal efferent activities observed in humans and experimental animals.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Anesthetics impair the homeostatic mechanisms involved in reflex regulation of cardiovascular function. The effects of volatile anesthetics on the baroreflex have been examined by measuring the bradycardic response of the heart during activation of baroreceptors in humans and experimental animals (1–3). These studies indicated that the attenuation of reflex bradycardia by volatile anesthetics is due to depression of parasympathetic outflow and increased sympathetic activity.

To examine the postsynaptic mechanisms of general anesthetics on vagal cardiac control, we isolated the cardiac neurons from the interatrial septum of bullfrog hearts and directly examined the effects of volatile anesthetics on the Ca2+ current of the parasympathetic neurons for the first time. The enzymatically isolated parasympathetic neurons in the ganglia of bullfrog show spherical cell bodies without dendrites and are suitable preparations for the voltage-clamp method and the analysis of activation/inactivation kinetics because of minimal interference due to membrane capacitance currents. Because only one type of neuron is present in the bullfrog interatrial septum (4), the intracardiac parasympathetic neuron is an accessible preparation for physiological and pharmacological studies.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
All solutions were made with 18 M{Omega} purity water and Analar grade chemicals and were saturated with 100% oxygen. The solutions used in the current study were: (a) Ringer's solution (in mmol/L): NaCl 110.0, KCl 2.5, MgCl2 5.0, CaCl2 2.5, HEPES 5.0, glucose 10.0 (pH adjusted to 7.4 with 1 mol/L NaOH); (b) Na+, K+-free solution: N-methyl-D-glucamine 110.0, MgCl2 5.0, CaCl2 2.5, HEPES 5.0, glucose 10.0 (pH adjusted to 7.4 with 2 mol/L HCl); and (c) internal solution: aspartic acid 80.0, tetraethylammonium chloride 20, adenosine 5'-triphosphate 2.0, HEPES 10.0, EGTA 5.0 (pH adjusted to 7.2 with 1 mol/L CsOH). The Ca2+-free Ringer's solution was identical to the Ringer's solution except that CaCl2 was omitted. Isoflurane and sevoflurane were generously donated from Dinabot Co. Ltd. (Osaka, Japan) and Maruishi Pharmaceutical Co. (Osaka, Japan), respectively.

The technique we used to isolate and culture parasympathetic neurons is a modification of a method previously described (4). After obtaining approval from our animal care committee, adult bullfrogs (Rana catesbeiana) were killed by decapitation under ether anesthesia, and their hearts were removed. The interatrial septum was dissected under the Ringer's solution, and the vagus nerve trunks were removed and cut into 1- to 2-mm pieces. The segments of vagus nerve were incubated in the Ca2+-free Ringer's solution containing 0.2% collagenase (type I; Sigma Chemical Company, St. Louis, MO) and 0.1% elastase (type III; Sigma) for 40 min, then 0.15% trypsin (type III-S; Sigma) was added to the solution. The parasympathetic neurons were dispersed by gently triturating the nerve segments with a Pasteur pipette. The dissociated tissue was plated onto culture dishes whose bottoms were coated with concanavalin-A (Sigma). The neurons were maintained in a tissue culture medium consisting of Leibovitz's L-15 medium (Gibco, Grand Island, NY). The neurons were incubated in a refrigerator at 4°C and were used 1–7 days after plating.

A tissue culture dish was mounted on the stage of an inverted microscope. The stage was specifically designed for the purpose of mounting the culture dish and for superfusion of the solutions in situ. The Na+, K+-free solution was continuously superfused through the dish at a rate of 1 mL/min during experiments. A patch-clamp technique (5) was used for the whole-cell voltage-clamp recordings. Electrodes were made from 1.0-mm inner diameter borosilicate capillary tubing and the electrode resistance was 1–5 M{Omega} when filled with the internal solution. Type EPC-7 amplifier (List-Medical, Darmstadt, Germany) was used for voltage-clamp recordings and corrections for liquid junction potentials between external and internal solutions (6). The cell membrane capacitance was estimated from the compensation dial settings on the amplifier. Membrane currents were monitored on a storage oscilloscope and were digitized simultaneously (14.4-kHz, 12-bit resolution) using a MacADIOS (GW, Somerville, MA) A/D converter board and were stored on a Macintosh computer (Apple, Cupertino, CA). A scientific graphing software (SigmaPlot; Jandel, San Rafael, CA) was used for analysis, curve-fits, and plots of the digitized data. The voltage-dependent Ca2+ current was isolated from the other membrane conductances in parasympathetic neurons after blocking Na+ and K+ currents using Na+- and K+-free internal/external solutions (4). The Ca2+ current was recorded in response to 200-ms depolarizing pulses from a holding potential of -80 mV. The inactivation time course of bullfrog parasympathetic neurons is not modified by substituting Ca2+ with Ba2+, which indicates that Ca2+-dependent inactivation (7) is minimal in these neurons (4). Additionally, the intracellular Ca2+ was buffered with EGTA in the current experiments. All experiments were performed at room temperature (20–22°C).

Isoflurane and sevoflurane were vaporized with a isoflurane vaporizer (Muraco, Tokyo, Japan) and a sevoflurane vaporizer (Ohmeda, West Yorkshire, UK), respectively, in 100% oxygen (1 L/min), then bubbled into the Na+, K+-free solution in a reservoir for at least 5 min before application to cells. Anesthetic concentrations are presented as volume percentage (vol%) in 100% oxygen. The concentrations of isoflurane and sevoflurane in the gas phase were verified by using an anesthetic gas analyzer (Capnomac; Datex, Helsinki, Finland), and the concentrations in solution were analyzed by using gas chromatography. The concentrations of isoflurane and sevoflurane in solution were found to be linear (0.64 and 0.55 mmol/L per 1%, respectively) with the percentage in gas phase up to 5.0%. A local perfusion technique was used for first application of anesthetics (8). Capillary tubing (tip diameters 100–200 µm) was mounted on a right-angled holder connected to a 10-mL reservoir, which contained the anesthetic-equilibrated solution. During administration of anesthetics, the capillary tubing was positioned near the cell, and anesthetic solutions in the reservoir flowed down to the recording dish by gravity. Because the diameter of the tip of the capillary tubing was several times bigger than the diameter of the neurons, and because the capillary was positioned upstream from the direction of the perfusate, the concentration of anesthetics exposed to the neurons was equivalent to that contained in the reservoir. Experimental observations under these conditions were performed after 3 min of exposure to the anesthetics, which was sufficient to produce stable effects. Data were analyzed only if complete recovery (90%–110% of control) occurred on washout of anesthetics.

Values are expressed as mean ± SD. Differences among multiple groups were tested by using analysis of variance, and differences between paired sets of data were compared by using a paired t-test. P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Examples of the effects of isoflurane (5.0 vol%) on the Ca2+ currents are shown in Figure 1A. The Ca2+ current was elicited with a 200-ms depolarizing pulse to 10 mV from a holding potential of -80 mV after blocking Na+ and K+ currents. The Ca2+ current was activated within 10 ms, then inactivated very slowly during the depolarizing pulse. Isoflurane reduced the amplitude of Ca2+ currents in a reversible manner. Figure 1B shows the current-voltage relations in the presence and the absence of isoflurane (2.5, 5.0 vol%). The protocol consisted of a series of 200-ms clamp pulses between -70 and 70 mV from a holding potential of -80 mV. The peak Ca2+ currents were normalized to the capacitance of each cell. The amplitude of Ca2+ current increased with depolarization (peak at 10 mV), decreased with further depolarization, and reversed direction (reversal potential at 60 mV). Isoflurane (2.5, 5.0 vol%) and sevoflurane (5.0 vol%) significantly reduced the peak Ca2+ currents (at 10 mV) to 79%, 53%, and 72% of control, respectively, without changing the reversal potentials.



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  		Figure 1. A, Representative recordings of the effects of isoflurane (5.0 vol%) on the Ca2+ currents of parasympathetic neurons. The currents were elicited in response to a 200-ms depolarizing pulse to 10 mV from a holding potential of -80 mV. C = control, I = isoflurane, W = recovery response on washout. B, The effects of isoflurane (2.5 vol%, n = 8; 5.0 vol%, n = 7) on the current-voltage relationships were obtained by plotting the peak normalized Ca2+ currents (cell membrane current/cell membrane capacitance) as a function of the membrane potential. {circ} = control, • = 2.5 vol% isoflurane, {blacktriangleup} = 5.0 vol%. Symbols represent mean ± SD. Each isoflurane curve significantly (P < 0.05) differs from the control curve as assessed by two-way analysis of variance.

 
The curve-fit analysis revealed that the inactivation process of the Ca2+ current consisted of at least three components (fast and slow inactivating currents and constant current) and was described by double exponential equation of the form: Go


where t is the time (s) after a peak of the Ca2+ current; A1, A2, and C are the fast, slow, and constant components of amplitude, respectively; and {tau}f1 and {tau}f2 are the fast and slow components of time constant, respectively (9). Both isoflurane and sevoflurane reduced A2 and {tau}f2 without a significant change in A1, {tau}f1, and C, except that a high concentration of isoflurane (5.0 vol%) significantly reduced C (Fig. 2, Table 1). The results indicate that volatile anesthetics accelerate inactivation of the slow component of Ca2+ current.



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  		Figure 2. Examples of the time courses of inactivation in the Ca2+ currents in the absence ({circ}) and the presence (•) of isoflurane (5.0 vol%). The decay of the currents were well fitted by a double-exponential time course. The continuous lines are described by Equation 1.

 

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  		Table 1. Effects of Volatile Anesthetics on Variables of the Ca2+ Current in Cardiac Neurons
 
We further evaluated the steady-state inactivation of the Ca2+ current in the absence and presence of a volatile anesthetic (Fig. 3). After a prepulse (5–10 s) between -80 and 10 mV (to inactivate the Ca2+ current), a 200-ms test pulse to 10 mV elicited the Ca2+ current whose peak depends on the prepulse potential (Fig. 3A). The ratio for the Ca2+ current per maximal current (I/Imax) was plotted as a function of the prepulse potential (Fig. 3B). The results were fitted to a Boltzman function of the form:


where Vm is the pre-pulse potential, V1/2 is the midpoint potential, and k is the slope factor. The control values of V1/2 and k were -17.6 ± 4.5 mV and 11.9 ± 1.3, respectively. Isoflurane (2.5 vol% and 5.0 vol%) significantly shifted the V1/2 toward the negative, to -31.9 ± 5.8 (n = 5) and -38.1 ± 10.0 (n = 4) mV, respectively, without changing k values, which indicates volatile anesthetic enhancement in the probability of closing the inactivation gate of the Ca2+ channel.



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  		Figure 3. Effects of isoflurane (2.5 and 5.0 vol%) on the steady-state inactivation of the Ca2+ currents. A, The Ca2+ currents were elicited with a test pulse to 10 mV after prepulses of -80, -60, -40, -20, and 0 mV in the absence (a) and presence (b) of isoflurane (2.5 vol%). B, The ratio for the Ca2+ current per the maximal current (I/Imax) was plotted. {circ} = control, • = 2.5 vol% isoflurane, {triangledown} = 5.0 vol%. The continuous lines are the best fit values to Equation 2. Symbols represent mean ± SD. n = 6. Each curve significantly (P < 0.05) differs from the control curve as assessed by two-way analysis of variance. An example of the double-pulse protocol is shown in the inset.

 
We did not analyze the activation kinetics because the interference caused by the membrane capacitance could not completely compensate. However, the times to peak were not significantly altered by the administration of isoflurane and sevoflurane (Table 1), which suggests minimal effects on the Ca2+ current activation.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The parasympathetic nervous system regulates cardiac functions. Some authors have suggested that the parasympathetic system has a protective role in myocardial injury and a prognostic role in outcome after cardiac and neurological injury (10,11). Although volatile anesthetics depress the vagal efferent activity in vivo, few studies have examined the effects of general anesthetics on the parasympathetic ganglionic activity (1,3). It is difficult to directly investigate the effects of general anesthetics on resting levels of ganglionic outflow in vivo because the cardiovascular and/or respiratory systems must be held constant during anesthetic administration to exclude the contributions of indirect actions to the preganglionic activity.

Although in vitro studies are generally limited by species specificity, temperature differences, and a lack of blood-borne factors from perfusate, direct anesthetic actions on ganglionic activity can be measured in isolated single parasympathetic neurons. In the voltage-clamp study, we demonstrated the volatile anesthetic inhibition of the Ca2+ current in the intracardiac parasympathetic ganglia. The voltage-dependent Ca2+ currents in general are involved in the release of neurotransmitters from nerve terminals (12). Parasympathetic neurons in the intracardiac ganglia release acetylcholine (Ach) during vagal stimulation and exert negative chronotropic effects on myocardium via activation of muscarinic Ach receptors (13). Because inhibition of Ca2+ currents reduce neurotransmitter release from neurons, the volatile anesthetic depression of the Ca2+ current in parasympathetic neurons could lead to a reduction of Ach release from the postganglionic endings and attenuation of vagal efferent activity on myocardium.

In this study, we demonstrated that volatile anesthetics accelerate Ca2+ current inactivation and shift the steady-state inactivation curve to the negative. Our results also indicate the increase in probability of closing the inactivation gate of the Ca2+ channel. Two studies (14,15) reported the acceleration of Ca2+ current inactivation by isoflurane in hippocampal neurons and ventricular myocytes, respectively, and Hirota et al. (16) determined that the volatile anesthetic depression of myocardial Ca2+ current is mainly a result of the speeding of the inactivation. Thus, modulation of the channel inactivation might be one of the main mechanisms of volatile anesthetic inhibition of the Ca2+ current. A high concentration of isoflurane (5.0 vol%), however, decreased the constant component of Ca2+ current, which suggests that additional mechanisms (e.g., decrease of the channel conductance to Ca2+) might be involved, as previously proposed (16).

Several types of Ca2+ channels are present in neurons, and separation of the components of Ca2+ currents are based on different sensitivity to various drugs (17). Isoflurane inhibits multiple Ca2+ channels in hippocampal pyramidal neurons (14). We did not study the individual Ca2+ channel subtypes because our primary objective was to reveal the effects of volatile anesthetics on the native Ca2+ current of parasympathetic neurons. The Ca2+ channel responsible for neurotransmitter release in the parasympathetic nerve ending is the N-type channel (18). Further experiments on the channels subtypes are required to argue for the anesthetic depression of Ach release.

In conclusion, isoflurane and sevoflurane reduced the amplitude of Ca2+ currents and modified the inactivation kinetics of the currents in parasympathetic neurons from bullfrog hearts. The results suggest that parasympatholytic actions after volatile anesthetic application are due to depression of the Ca2+ current in parasympathetic neurons.


   Acknowledgments
 
Supported by the Ministry of Education, Science and Culture, Japan.

We thank Dr. K. Toriizuka for performing assays of isoflurane and sevoflurane bath concentrations.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication March 9, 1999.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press