Volatile anesthetics, such as halothane and isoflurane, have been reported to affect the endothelium mediated relaxation of vascular smooth muscle cells. Because the activity of the constitutive nitric oxide synthase in endothelial cells depends on the availability of intracellular Ca2+, there is a definite possibility that the observed inhibitory effect of volatile anesthetics involves an action on the agonist-evoked internal Ca2+ mobilization and/or Ca2+ influx in these cells. Therefore, a study was undertaken to determine how halothane and isoflurane affect the Ca2+ signalling process in vascular endothelial cells.
The effect of halothane and isoflurane on the Ca2+ response to bradykinin of bovine aortic endothelial (BAE) cells was investigated using the fluorescent Ca2+ indicator fura-2. Halothane or isoflurane was applied either to resting cells or after bradykinin stimulation. The agonist-evoked Ca2+ influx in BAE cells was estimated by measuring either the rate of fura-2 quenching induced by Mn2+ or the increase in cytosolic Ca2+ concentration initiated after readmission of external Ca2+ after a brief exposure of the cells to a Ca(2+)-free external medium. The effects of halothane on cell potential and intracellular Ca2+ concentration were measured in cell-attached patch-clamp experiments in which a calcium-activated K+ channel and an inward rectifying Ca(2+)-independent K+ channel were used as probes to simultaneously monitor the intracellular Ca2+ concentration and the cell transmembrane potential. In addition, combined fura-2 and patch-clamp cell-attached recordings were carried out, to correlate the variations in internal Ca2+ caused by halothane and the activity of the Ca(2+)-dependent K+ channels, which are known in BAE cells to regulate intracellular potential. Finally, a direct action of halothane and isoflurane on the gating properties of the Ca(2+)-activated K+ channel present in these cells was investigated in patch-excised inside-out experiments.
The results of the current study indicate that the initial Ca2+ increase in response to bradykinin stimulation is not affected by halothane, but that pulse applications of halothane (0.4-2 mM) or isoflurane (0.5-1 mM) reversibly reduce the sustained cytosolic Ca2+ increase initiated either by bradykinin or by the Ca2+ pump inhibitor thapsigargin. In addition, halothane appeared to dose-dependently inhibit the Ca2+ influx evoked by bradykinin, and to cause, concomitant to a decrease in cytosolic Ca2+ concentration, a depolarization of the cell potential. Halothane failed, however, to affect internal Ca2+ concentration in thapsigargin-treated endothelial cells, which were depolarized using a high K+ external solution. Finally, halothane and isoflurane decreased the open probability of the Ca(2+)-dependent K+ channel present in these cells.
These observations suggest that the effects of halothane and isoflurane on Ca2+ homeostasis in BAE cells reflect, for the most part, a reduction of the thapsigargin- or bradykinin-evoked Ca2+ influx, which would be consequent to a cellular depolarization caused by an inhibition of the Ca(2+)-dependent K+ channel activity initiated after cell stimulation.
VOLATILE anesthetics such as halothane and isoflurane have been reported to have specific and differing cardiovascular effects. These effects are likely to involve several mechanisms acting on the integration of factors that control smooth muscle cell contraction. It is widely accepted that the endothelium constitutes a key regulator of vascular tone by secreting both vasoconstrictors, such as endothelin-1 (and perhaps other endothelins), and vasorelaxing agents, such as the prostacyclin PGI2and the major endothelium-derived relaxing factor nitric oxide (NO). The effects of anesthetics on the endothelium-dependent control of vascular tone were examined in several studies. For instance, Blaise et al. showed that isoflurane impairs the phenylephrine-induced contractile response of canine coronary arteries in an endothelium-dependent manner. It was suggested that isoflurane could, in this case, stimulate the release of NO. However, most studies supported an inhibitory action of volatile anesthetics on the NO-evoked vascular relaxation. Halothane was found, by Muldoon et al., to inhibit the endothelium-dependent vasodilation induced by acetylcholine and bradykinin in isolated contracted rabbit and canine vascular rings. In addition, Uggeri et al.'s study on rat thoracic aorta provided evidence that halothane, enflurane, and isoflurane attenuate the NO-dependent relaxation induced by muscarinic receptor stimulation. Finally, Toda et al. confirmed that halothane and isoflurane inhibit both the endothelium-dependent relaxation and the cyclic guanosine monophosphate formation elicited by acetylcholine in rat aorta. On the basis of these observations, it was concluded that halothane and other volatile anesthetics affect either the production and release of NO [4,5]or its stability. Most studies failed to show an effect that could be attributable to a direct action on vascular smooth muscle cells that involve guanylate cyclase, although Hart et al. reported, in a work on rat aorta, a series of results suggesting that the site of action of halothane was at the vascular smooth muscle level.
Studies have indicated that the production and release of NO after the stimulation of endothelial cells by bradykinin, histamine, adenosine triphosphate, substance P, or thrombin are linked to an increase in intracellular Calcium2+. [9–12]In fact, Calcium2+is known to activate, with calmodulin and the nicotinamide adenine dinucleotide NADH, an NO synthase that, in turn, metabolizes L-arginine to citrulline and NO. [10,13]In addition, there is strong evidence that the increase in intracellular Calcium2+caused by vasodilators such as bradykinin, adenosine triphosphate, and histamine is a biphasic process that reflects, in part, an inositol 1,4,5-triphosphate (InsP3)-mediated release of Calcium2+from intracellular stores, coupled to a Calcium2+entry from the external medium. [14–16]The molecular mechanism underlying the agonist-evoked Calcium2+influx in endothelial cells remains ill defined.
Electrophysiological and unidirectional45Calcium2+flux measurements in cultured or freshly dissociated endothelial cells from large blood vessels have indicated an absence of depolarization-activated Calcium2+influx via voltage-dependent Calcium2+channels in these cells. As in lymphocytes and certain other nonexcitable tissues, there is clear evidence that the Calcium2+influx in endothelial cells is augmented or stabilized at hyperpolarizing potentials. [16,18,19]It is generally agreed that this augmentation arises from the increased electrical driving force acting on Calcium2+ions under these conditions. [17,19]This situation is at variance with the results obtained in excitable cells where Calcium2+influx is normally reduced at hyperpolarizing potentials because of the closure of voltage-dependent Calcium2+channels. A hyperpolarizing response to cell stimulation may result from several factors, but there is increasing data that support a determinant role of the Potassium+selective channels in the case of endothelial cells. [17,19,20]In particular, studies have shown that Calcium2+-activated Potassium (+) channels [Potassium(Calcium2+)] constitute important positive feedback elements of the Calcium2+signalling process in vascular endothelial cells. [16,18,19]Chemical agents that affect Potassium(Calcium2+) channel activity are, therefore, likely to modulate the agonist-evoked Calcium2+influx in these cells.
Because volatile anesthetics were shown to alter internal Calcium2+homeostasis in many cell types, [21,22]including endothelial cells, there is a possibility that the reported inhibition by halothane or isoflurane of the endothelium-dependent relaxation of vascular endothelial cells involves an action of the anesthetics on the InsP3-dependent Calcium2+mobilization and/or agonist-evoked Calcium2+influx. Fura-2 and patch-clamp experiments, therefore, were undertaken to characterize the effects of halothane and isoflurane on the Calcium2+response of bovine aortic endothelial (BAE) cells to the vasodilating agent bradykinin and to determine the involvement of the endothelial cell Potassium(Calcium2+) channels in this process. Our results indicate that the action of halothane includes an inhibition of the agonist-evoked Calcium2+influx, which is partly related to depolarization of the endothelial cell potential that results from an inhibition of the Potassium(Calcium2+) channels present in these cells.
Materials and Methods
The details of the BAE cell culture procedure and characterization have been described elsewhere. The cells were tested with endothelial cell-labelling reagents or factor VIII antibodies (Daco, Santa Barbara, CA) and responded positively. Bovine aortic endothelial cells were cultured in Dulbecco's modified Eagle Medium (Gibco, Gaithersburg, MD) supplemented with 10% newborn calf serum, 3.7 g/l NaHCO (3), 100 U/ml penicillin, and 100 micro gram/ml streptomycin in humidified air, with 5% CO2atmosphere at 37 degrees C. Cells from serial passage 21–26 were reseeded on microscope cover slips, to accommodate the superfusion chamber used for fluorescence and patch clamp measurements.
Solutions and Drugs
Confluent cells were superfused continuously with an Earle's solution that had the standard composition, as follows (in mM): 121.0 NaCl; 5.4 KCl; 1.8 CaCl2; 0.8 MgSO4; 6.0 NaHCO3, 1.0 NaH2PO4; 5.5 glucose buffered at pH 7.3 with 25.0 Hepes, and 10.0 NaOH. Potassium+-Earle's solutions were prepared by equimolar substitution of sodium chloride and NaOH by KCl and potassium hydroxide. Calcium2+-free (0 Calcium2+) solutions were prepared by omitting CaCl2and adding 1 mM ethyleneglycol-bis-[B-aminoethylether]N,N,N',N' (tetraacetic acid [EGTA]) to standard Earle's or Potassium+-Earle's solutions.
The low-Calcium2+solution used to calibrate the fura-2 signal consisted of an Earle's medium with no CaCl2, to which was added 5.0 mM EGTA, 15.0 mM NH4Cl, and 5 micro Meter ionomycin. The high-Calcium2+solution used for calibration was an Earle's solution, with 5 micro Meter ionomycin buffered at pH 8.0–8.5. Patch pipettes were filled with a solution that contained (in mM): 200 KCl, 0.5 MgSO4, 0.91 CaCl2, and 1 EGTA, for a free Calcium2+concentration of 1 micro Meter.
For whole cell recordings, the free Calcium2+concentration was reduced to 0.2 micro Meter. The pH was buffered at 7.3, KOH with 25 mM Hepes and 10 mM. Bradykinin, ionomycin, and fura-2 were purchased from Sigma (St. Louis, MO); thapsigargin was obtained from L.C. Services (Woburn, MA). Halothane and isoflurane (Ayerst, Montreal, Quebec) containing solutions were prepared in 50-ml gas-tight syringes (Hamilton 1050, Reno, NV), at concentrations ranging from 0.4 mM to 2 mM (2–10 micro liter halothane in 50 ml solution) or 0.5 mM to 1 mM (3.8–7.9 micro liter isoflurane in 50 ml solution), respectively. As discussed by Franks and Lieb, a concentration of 1 mM halothane corresponds at room temperature (25 degrees C) to a gaseous partial pressure of 1.7 volume percent (1.7%), whereas 1 mM isoflurane is equal to a partial pressure of 2.0 volume percent (2.0%). At this temperature, the EC50of halothane for general anesthesia ranges from 0.17 mM to 0.24 mM and from 0.23 mM to 0.25 mM for isoflurane. The syringes were mounted on perfusion pumps (Harvard 11, South Natick, MA) set at a perfusion rate of 2–4 ml/min. The effective halothane or isoflurane concentration in the perfusion chamber was estimated as described previously by gas chromatography. .
Confluent monolayers of BAE cells were loaded with fura-2 by incubation in an Earle's solution that contained 3 micro Meter fura-2 acetoxymethyl ester (fura-2 AM). The incubation time ranged from 30 min to 40 min at room temperature. The fluorescence from 15–20 cells was measured with a Nikon inverted microscope (Tokyo, Japan) attached to a dual-excitation spectrofluorometer (Spex Fluorolog II, Edison, NJ) with excitation wavelengths set at 350 nm and 380 nm, respectively. A dichroic mirror (Nikon FT 400) was placed in the excitation pathway and the emission monitored at 500 nm with a standard bandpass filter (Andover Corporation 500FS40, Salem, NH). The cytosolic Calcium2+concentration was calculated from the ratio of the fluorescence measured at 350 nm and 380 nm, respectively, and from the ratio of fluorescence at 380 nm in low-Calcium2+relative to that in high-Calcium2+conditions, as described previously. For experiments in which Manganese2+was used as a quenching agent, one excitation wavelength was set at 358 nm, to monitor fura-2 fluorescence independently of the intracellular Calcium2+concentration. The rate of quenching of the fura-2 signal was calculated as follows:Equation 1where cps is the proton count per second, RLF[Manganese2+] is the rate at which the fura-2 fluorescence intensity decreased calculated over the first 30 s after the addition of Manganese2+(period during which the decrease of fluorescence remains linear), and RLF the equivalent rate calculated during a 60-s period before the addition of Manganese2+to the external medium (this measure reflects the leak of fura-2 out of the cells). All the experiments were performed at room temperature (23–25 degrees C).
Patch pipettes were pulled from Pyrex capillaries (Corning 7040, Corning, NY), using a David Kopf (Twinga, CA) programmable pipette puller (Model 750) and used uncoated. The resistance of the patch electrode ranged from 4 to 10 M Omega. Current traces were recorded on frequency modulator wideband tapes (HP 3964, San Diego, CA) at a bandwidth of 1.25 kHz and subsequently transferred to a PC hard disk for offline analysis. Unless otherwise specified, the signal was sampled at 1.5 kHz and filtered at 500 Hz, with two low-pass four-pole Bessel filters (VVS 300B, Frequency Devices, Haverhill, MA) connected in series. Baseline drift was corrected through a multiple linear interpolation procedure. The open channel probability was computed from current amplitude histograms on the basis of a binomial distribution as described elsewhere. The current amplitude histograms were computed from current segments of 60 s minimum duration. Results are expressed as means+/-SEM.
(Figure 1) shows the effect on halothane on the cytosolic Calcium2+concentration after bradykinin receptor stimulation. Panel A illustrates the Calcium2+response evoked by bradykinin in control conditions (n = 14) and in the presence of 2 mM halothane (n = 5). The initial Calcium2+rise related to the release of Calcium2+from intracellular stores appeared unaffected by halothane at concentrations ranging from 0.2 mM to 2 mM, with a peak Calcium2+value estimated at 700+/-300 nM (n = 5) in control and 700+/-250 nM (n = 5) in halothane conditions. Identical results were obtained using cells preincubated for 5 min in halothane (2 mM; data not shown). In cells exposed to halothane, however, there was a clear increase in the rate at which the Calcium2+concentration returned to a stable resting value. Computation of t1/2, the time needed for the Calcium2+concentration to reach a value equal to half the initial Calcium2+peak amplitude, yielded values of 148+/-45 s (n = 5) in control and 81+/-24 s (n = 5) in 2 mM halothane conditions, respectively.
An example of the Calcium2+response initiated after the withdrawal of 0.8 mM external halothane is presented in panel B. This experimental maneuver typically produced a transient Calcium2+increase, which decayed to a Calcium2+concentration value higher than the Calcium2+level prevailing before halothane withdrawal. Panel C shows the effects of brief, 50-s halothane applications (2 mM; n = 12) on the Calcium2+response triggered by bradykinin. Under these conditions, halothane appeared to significantly decrease the internal Calcium2+concentration, especially during the plateau phase of the Calcium2+response.
In 2 (16%) of 12 of the experiments performed in 2 mM halothane, the concentration of Calcium2+decreased rapidly, after an initial Calcium2+increase, and halothane was ineffective in further reducing the Calcium2+concentration. In the remaining experiments (10 of 12), 2 mM halothane caused an average reduction of the Calcium2+concentration of 145+/-35 nM (n = 10), for a relative 70% decrease of the Calcium2+level maintained during the plateau phase. In addition, each withdrawal of halothane was followed by a transient Calcium2+increase, confirming the Calcium2+response illustrated in Figure 1(B). However, because halothane was reapplied during the onset of the Calcium2+rise, the Calcium2+transients in this case decayed to a value close to the resting Calcium2+concentration before bradykinin stimulation.
A similar Calcium2+response was observed using isoflurane at concentrations of 0.5 mM and 1 mM, suggesting that both anesthetics affect Calcium2+signalling in endothelial cells through a common mechanism (panel D). The average reduction in Calcium2+concentration was estimated at 247+/-130 nM (n = 8) and 250+/-100 nM (n = 9) at 1 mM and 0.5 mM isoflurane, respectively. As observed with halothane, isoflurane appeared ineffective in reducing the initial Calcium (2+) rise after bradykinin receptor stimulation (n = 10; data not shown). These observations indicate that the effects of halothane and isoflurane influence the external Calcium2+-dependent phase of the Calcium2+increase evoked by bradykinin, rather than the initial Calcium2+increase, which is due to the mobilization of Calcium2+from intracellular Calcium2+stores.
A series of experiments was performed in which the endoplasmic Calcium2+pump inhibitor thapsigargin was used to initiate a release of Calcium2+from internal pools and generate a capacitative Calcium (2+) influx, independent of InsP3production. [28,29] Figure 2shows the effects of 2 mM halothane when applied for time intervals of 50 s to 100 s after 500 nM thapsigargin stimulation (n = 5). A reduction of 50+/-25 nM (n = 5) of the Calcium2+increase caused by thapsigargin was observed under these conditions, indicating that halothane could still affect the cytosolic Calcium2+concentration despite the absence of InsP3production. As in Figure 1(C and D), the withdrawal of halothane resulted in transient Calcium2+rises, with peaks that reached, in this case, concentration values superior to the Calcium2+concentration measured after thapsigargin application. The fura-2 measurements presented in Figure 2do not support an action of halothane based exclusively on inhibition of the InsP3production machinery.
One possible explanation for the observed effects of halothane on thapsigargin-treated cells would be an inhibition of the capacitative Calcium2+influx known to be secondarily activated by emptying internal Calcium2+stores in these cells. [28–31]Experiments were performed in which the effect of halothane was studied under InsP3-sensitive Calcium2+pool reloading conditions. The perfusion protocol that was used in this case is illustrated in Figure 3(A). Calcium2+was first released from the intracellular InsP3-sensitive stores by a double application of 10 nM bradykinin in Earle's Calcium2+-free solution. After the second application of bradykinin, the bath solution was replaced by a Calcium2+-free Earle's medium with 2mM halothane. The agonist-evoked Calcium2+influx was then assayed by superfusing the cells for 50 s with a standard Earle's solution (1.8 mM CaCl2), with and then without 2mM halothane. The fura-2 recording presented in Figure 3(A) indicates a clear decrease of the resulting Calcium2+rise under halothane conditions. Such a behavior was observed in three additional experiments based on the same perfusion protocol. These results, therefore, would support a model in which halothane acts through an inhibition of the capacitative Calcium2+entry in these cells. This conclusion is also in agreement with the fura-2 experiment illustrated in Figure 3(B). The perfusion protocol used in this case is similar to the one presented in Figure 3(A), except that Calcium2+reloading was carried out either in the presence or in the absence of halothane. The amount of Calcium2+sequestered in the InsP (3)-sensitive Calcium2+pools during the reloading period was assayed by a third application of bradykinin in Calcium2+-free conditions. These experiments indicated that the release of Calcium2+after reloading in the absence of halothane was always larger than that measured when the reloading was carried out in the presence of halothane in the bathing medium. This observation provides evidence that the decrease in cytosolic Calcium2+initiated by halothane cannot be attributed to the stimulation of a Calcium2+sequestration process.
Evidence of an inhibitory action of halothane on the agonist-evoked Calcium2+influx in BAE cells was also obtained from fura-2 experiments in which Manganese2+was used as a quenching agent. Previous studies established that, as internal Calcium2+stores release Calcium2+, entry of extracellular Calcium2+and Manganese2+in endothelial cells is enhanced. The bradykinin-evoked Calcium2+influx in BAE cells was thereby estimated by measuring the rate at which 1 mM Manganese2+in the external medium induced quenching of the intracellular fura-2 signal in the presence and/or absence of halothane. Figure 4(A) shows that the fluorescence intensity at 358 nm remained unchanged, as expected, after bradykinin stimulation in Calcium2+-free external conditions. However, a clear decrease in fura-2 fluorescence was initiated by superfusing the cells with a solution that contained both Calcium2+(1.8 mM) and Manganese2+(1 mM). In addition, the rate of fura-2 quenching was lower in the presence of halothane in the external medium, indicating an inhibition of the Calcium2+influx pathway under these conditions. Figure 4(B) summarizes the results obtained at 0.8 mM and 2 mM halothane on four different cell preparations. A significant level of inhibition (42–64%) was observed with 2 mM halothane for all the cell preparations considered, but halothane at a concentration of 0.8 mM appeared effective in only two of the cell preparations with inhibition of 41%(preparation 4) and 47%(preparation 2), respectively. In the remaining two cell preparations, halothane was ineffective at this concentration. Experiments carried out on cell preparation 2 using 0.4 mM halothane yielded a mean rate of fura-2 quenching of 1910+/-470 cps/s (n = 4), for an inhibition of 24%. There was, however, large cell-to-cell variation between the responses of individual cells at this low concentration.
To test the hypothesis that part of the observed inhibition by halothane of the Calcium2+influx involves a depolarization of the cell potential, fura-2 experiments were performed in which the effect of halothane was investigated on BAE cells that were depolarized using a Potassium+-Earle's solution as bathing medium. Figure 5(A) shows an example of fluorescence measurements performed according to a perfusion protocol similar to the one presented in Figure 3(A). In contrast to the findings illustrated in Figure 3(A), the removal of halothane under these conditions failed to induce an increase of the cytosolic Calcium2+concentration (n = 9), despite evidence for a Calcium2+influx after Calcium2+readmission. A significant increase in cytosolic Calcium2+concentration could be observed, however, after the replacement, at the end of the superfusion protocol, of the Potassium+-Earle's bathing medium by a standard Earle's solution (E).
In additional experiments, halothane was applied to TG-treated cells under both normal external Earle's conditions (E) and in the presence of a Potassium+-Earle's external medium. The fura-2 measurements presented in Figure 5(B) confirm that halothane applied to TG-treated cells under normal Earle's conditions causes a reversible decrease in cytosolic Calcium2+concentration, as shown previously in Figure 2. A significant decrease in internal Calcium2+concentration was also initiated after the superfusion of the TG-treated cells with Potassium+-Earle's medium. Under these conditions, 2 mM halothane failed to cause a decrease in cytosolic Calcium2+concentration, indicating that the action of halothane on intracellular Calcium2+can be significantly impaired by maintaining the cells in a depolarized state. The results in Figure 5(B) are representative of six other experiments carried out under the same conditions. In one experiment, however, a small decrease in cytosolic Calcium2+was observed after the superfusion of the cells with an external solution that contained 2 mM halothane. The amplitude of the Calcium2+decrease was significantly smaller than that measured under normal Earle's conditions, and the withdrawal of halothane did not result in a transient Calcium2+rise (data not shown).
To determine the effect of halothane on membrane potential, whole cell experiments were performed on mechanically dissociated BAE cells in zero current clamp conditions, using 200 mM KCl + 0.2 micro Meter Calcium2+filled patch electrodes (see Materials and Methods). An example of the resulting voltage changes is presented in Figure 6(A). As expected, the superfusion with an external solution containing 10 nM bradykinin caused an initial hyperpolarization of the cell potential, the magnitude of which ranged from -45 mV to -55 mV (mean -49+/-4 mV; n = 4). An external application of halothane under these conditions induced a significant cellular depolarization (39+/-5 mV, n = 4), which was reversible after halothane removal. These results would support a model in which the halothane-induced inhibition of the capacitative Calcium2+influx in BAE cells is partly mediated by an action of halothane on the cell potential.
The relation between external halothane, Calcium2+influx, and membrane potential in bradykinin-stimulated BAE cells was investigated next in a series of experiments in which two Potassium+selective channels were used as probes to simultaneously monitor the cell potential and the intracellular Calcium2+concentration. The membrane potential was estimated by measuring, in the cell-attached configuration with a KCl-filled patch pipette (see Materials and Methods), the amplitude of the unitary current jump resulting from the openings of a Calcium2+-insensitive inward rectifying IK1channel present in BAE cells. The rationale of this approach is that the effective potential applied across a patch membrane in the cell-attached configuration is equal to the cell potential minus the potential in the patch pipette. For instance, a pipette potential of 30 mV will result in a -70 mV potential difference across the membrane patch area if the cell potential is equal to -40 mV. In Figure 6(B), (iii) shows the current/voltage relation of the inward rectifying IK1channel measured in cell-attached, patch-clamp experiments with KCl-containing pipettes (see Materials and Methods) on BAE cells bathed in a Potassium+-Earle's solution, to abolish the contribution of the cell resting potential. .
Using this curve as reference, it is then possible, knowing the amplitude of the unitary IK1channel current jumps, to determine the magnitude of the potential acting on the IK1channel. Because the pipette potential is maintained at a constant known value, it follows that any variation of the IK1channel unitary current jump amplitude is a reflection of a variation in intracellular potential. Cell-attached experiments can, therefore, be used to directly monitor the cell potential during bradykinin stimulation, with no significant disturbance of the cytoplasmic medium. Similarly, the fluctuations in intracellular Calcium (2+) concentration in BAE cells can be estimated by monitoring the changes in activity of the Potassium(Calcium2+) channels present in the BAE cell plasma membrane. Unlike the Potassium(Calcium2+) channel of large conductance measured in a variety of excitable and nonexcitable cells, the Potassium(Calcium2+) channel identified in BAE cells exhibits inward rectification, with a slope conductance of 40 pS and 10 pS for inward and outward currents, respectively. In inside-out patch clamp experiments, this channel was activated at submicromolar cytosolic Calcium (2+) concentrations, and channel activity appeared voltage insensitive within the voltage range -100 mV to 0 mV. As a consequence, both IK1and Potassium(Calcium2+) constitute endogeneous probes that can be used to simultaneously record changes in cell potential and internal Calcium2+concentration.
(Figure 6)(B) shows an example of single channel recording in which both IK1and Potassium(Calcium2+) are present. Downward current deflections corresponding to spontaneous IK1channel long openings were observed before the first addition of halothane to the external medium. The pipette potential was maintained at 30 mV throughout, and the measured unitary current amplitude corresponded to 1.8 pA. Because a potential difference of 53 mV is known to be required to generate unitary current jumps of 1.8 pA amplitude, it was concluded in this case that the cell resting potential was equal to -23 mV. This value is in good agreement with the whole cell measurements reported by Mehrke et al. and the zero current clamp results in Figure 6(A).
It should also be apparent from this recording that the application of 2 mM halothane, before bradykinin stimulation, did not result in any significant changes in the current fluctuation pattern. A small decrease of the IK1unitary current amplitude from 1.8 pA to 1.5 pA could, nevertheless, be detected, an observation compatible with a 9-mV depolarization of the cell potential. In contrast, a subsequent superfusion with a solution that contained 10 nM bradykinin resulted in an increase of the IK1channel unitary current jump amplitude from 1.6 pA to 3.8 pA, indicating a 63-mV hyperpolarization of the cell potential, to a value close to -86 mV [see enlargement (i)]. In addition, the presence of bradykinin in the external medium caused the appearance of current bursts, characterized by rapid channel openings, superimposed on the IK1slow current fluctuations [see enlargement (i)]. These fast openings were due to the activation of Potassium(Calcium2+) channels in response to the bradykinin-evoked increase in cytosolic Calcium2+concentration. Under these conditions, the addition of halothane (2mM) to the bathing medium led to a marked reduction in Potassium(Calcium2+) channel activity concomitant to a decrease of the IK1channel unitary current jump amplitude to 2.4 pA [see enlargement (ii)]. These observations were confirmed in four identical experiments, which showed an average reduction in unitary current amplitude of 1.5+/-0.4 pA (n = 5), signifying an average halothane-induced cell depolarization of 47 +/-10 mV. These results demonstrate that the stimulation of BAE cells by bradykinin causes a transient internal Calcium2+rise coupled to a hyperpolarization of the cell potential, both inhibitable by halothane.
The effect of halothane on cytosolic Calcium2+and Potassium(Calcium2+) channel activity was also investigated in a series of fura-2+ cell-attached patch-clamp experiments (n = 4), in which the internal Calcium2+concentration and the activity of the Potassium(Calcium2+) channels were measured simultaneously. An example of a recording is presented in Figure 7. As expected, there is a close correlation between the absence of Potassium(Calcium2+) channel activity, a lower internal Calcium2+level, and the presence of halothane in the bathing medium. In addition, the fura-2 recording in Figure 7provides further evidence that the withdrawal of halothane from the external medium constitutes a triggering event for the generation of a transient Calcium2+rise, to which is associated an increased Potassium(Calcium2+) channel activity (see fura-2 signal at the end of the recording).
The results in Figure 7suggest that part of the observed membrane depolarization evoked by halothane may be due to a reduced Potassium(Calcium2+) channel activity related to a decreased cytosolic Calcium2+concentration. However, the effect of halothane on the Potassium(Calcium2+) channel activity may not be due exclusively to an indirect action on the cytosolic Calcium2+level. The hypothesis of a direct action of halothane on the Potassium(Calcium (2+)) channel in BAE cells was tested in inside-out patch-clamp experiments, where the Potassium(Calcium2+) channel activity was measured at various doses of halothane at a fixed cytosolic Calcium2+concentration (Figure 8(A)). Halothane, at concentrations ranging from 0.4 mM to 2 mM, caused a significant reduction of the Potassium(Calcium (2+)) channel open probability (Po), inhibiting channel activity by 96 +/-3%(n = 8) and by 65+/-14%(n = 8) after 40-s exposures to 2 mM (i) and 0.4 mM (ii) halothane, respectively. There was no detectable effect on the Potassium(Calcium2+) channel unitary current amplitude at either of these halothane concentrations. The inhibitory action of halothane appeared reversible in most experiments, but the recovery time varied, with values from less than 20 s to more than 10 min. Generally, complete channel inhibition could be obtained in less than 10 s with 2 mM halothane, whereas a minimum of 40 s was needed to observe a stable reduction in channel activity at 0.4 mM. Channel inhibition could also be observed in the presence of isoflurane (Figure 8(B)). Reductions of channel activity varying from 35% to 85%(mean 52 +/-16%; n = 6) were measured with 0.5 mM isoflurane (i). Similar values were obtained with 1 mM isoflurane (ii), in which a mean inhibition of 54+/-15%(n = 4) was observed (Figure 8). These two volatile anesthetics, therefore, constitute potent Potassium(Calcium2+) channel blockers.
Our data suggest that part of the effects exerted by halothane and isoflurane on the Calcium2+response of endothelial cells after bradykinin stimulation is related to a depolarization of the cell potential resulting from the inhibition of a Potassium(Calcium2+) channel. Through this mechanism, halothane would decrease the capacitative Calcium2+influx linked to the mobilization of internal Calcium2+pools by decreasing the electrochemical driving force acting on Calcium (2+) ions.
Effects of Halothane on Calcium2+Mobilization
In several studies, it was reported that halothane impairs Calcium (2+) retention in isolated cardiac sarcoplasmic reticulum vesicles by increasing passive Calcium2+efflux. [21,34]. This effect appeared to be linked to the activation by halothane of the sarcoplasmic reticulum Calcium2+release channel. However, despite evidence for the existence of functional caffeine-sensitive Calcium2+pools in BAE cells, [9,29]our experiments (data not shown) failed to detect any significant rise in cytosolic Calcium2+after bath application of halothane on resting cells. Our observations are, therefore, in accordance with those of Loeb et al., who studied the alteration by volatile anesthetics of Calcium2+mobilization in BAE cells. In addition, the fura-2 results in Figure 1(A) showed that the initial Calcium2+rise in response to bradykinin stimulation is not affected by halothane. This is at variance with the findings published by Loeb et al., who reported that Calcium2+transients elicited by brief applications of bradykinin were significantly inhibited by halothane but not isoflurane. It is clear, however, from Figure 2that the production of InsP3is not essential to the halothane-induced decrease in cytosolic Calcium2+concentration. This would be in agreement with the results of Kress et al. and Stern et al. on neuronal cells, which suggested that halothane at clinical concentrations (< 0.8 mM) does not affect the phosphatidyl inositol signalling pathway nor significantly depress the InsP3-induced Calcium2+mobilization. Nevertheless, it must be pointed out that, in the absence of direct measurements of the InsP3and InsP4levels in cells exposed to halothane, we cannot entirely rule out that this agent exerts part of its inhibitory action via the phosphatidyl inositol pathway. In addition, halothane has been reported to affect cyclic adenosine monophosphate production in several cell types, including platelets, and so the effects of halothane on InsP3production could also be mediated via an action on cyclic adenosine monophosphate.
Effect of Halothane or Isoflurane on Capacitative Calcium (2+) Influx: Role of Membrane Potential
Part of the experimental evidence in support of an effect of volatile anesthetics on the agonist-evoked Calcium2+influx in endothelial cells came from fura-2 recordings such as the ones illustrated in Figure 1(B, C, and D). In these experiments, halothane or isoflurane was applied during the secondary phase of the Calcium2+response of BAE cells to bradykinin stimulation. It is generally agreed that the sustained intracellular Calcium2+increase that follows the initial Calcium2+rise due to the release of Calcium2+from internal Calcium2+stores depends, to a large extent, on the entry of Calcium2+from the external medium. The fact that halothane and isoflurane caused a significant decrease of the cytosolic Calcium2+concentration during the external Calcium2+-dependent phase of the BAE cell response to bradykinin stimulation provides indirect support for an action of volatile anesthetics on the agonist-evoked Calcium2+entry mechanism in these cells.
More importantly, it is clear from the fura-2 measurements presented in Figure 1(B, C, D), Figure 2, and Figure 7that the withdrawal of halothane or isoflurane from the external medium led to transient Calcium2+increases whose peak values could exceed the Calcium2+level measured before the first exposure of the cell to the anesthetic (Figure 2). Similar transient changes in cytosolic Calcium (2+) concentration were reported for a variety of nonexcitable cells, including BAE cells, upon readmission of external Calcium2+after a short incubation in Calcium2+-free solution. [39,40]The transient Calcium2+increases measured under these conditions were usually interpreted as reflecting an entry of Calcium2+via a Calcium (2+)-permeable pathway activated by the decreased state of filling of the internal Calcium2+stores (capacitative influx). .
These results are also in agreement with the observation illustrated in Figure 3(A), in which the removal of halothane enhanced the Calcium2+rise initiated upon readmission of external Calcium2+after Calcium2+pool depletion. One likely explanation compatible with the results in Figure 1(B, C, D), Figure 2, and Figure 7would be that the withdrawal of halothane or isoflurane removed the volatile anesthetic-induced inhibition of the capacitative Calcium2+influx evoked either by bradykinin or TG. The exact mechanism underlying the time course of the transient peaks initiated after Calcium2+readmission or anesthetic removal is not yet fully resolved. One possibility would be that the Calcium2+entry is inactivated at high and reactivated at low intracellular Calcium2+. This would be in line with the observation of Vaca and Kunze, who reported that a Calcium2+-release activated Calcium2+current in BAE cells could only be recorded in the presence of very low intracellular Calcium2+.
In several studies, researchers indicated that the agonist-stimulated Calcium2+influx in several endothelial cell types, including BAE cells, is decreased in response to a cell depolarization. [18,19]The diminution in cytosolic Calcium2+concentration shown in Figure 5(B) after the substitution of a normal Earle's medium by a Potassium+-Earle's solution in TG-stimulated BAE cells fully supports this conclusion. Schilling reported that the Calcium2+influx, reflected by the amplitude of the transient Calcium (2+) increases triggered by readmission of external Calcium2+after exposure of bradykinin-stimulated BAE cells to a Calcium2+-free external solution, was reduced, but not abolished, in 150 mM external KCl. This would be in agreement with the results presented in Figure 5(A), where a significant increase in internal Calcium2+could still be detected after the replacement of a Calcium2+-free Potassium+-Earle's medium by a Calcium2+-containing Potassium+-Earle's solution. The internal Calcium2+augmentation observed under these conditions is expected to reflect a capacitative Calcium2+influx, because control experiments indicated no changes in cytosolic Calcium2+concentration after the replacement of a Potassium+-Earle's solution by a Calcium2+-free Potassium+-Earle's bathing medium in resting cells (data not shown). However, in contrast to the fura-2 recording presented in Figure 3(A), the removal of halothane in Potassium+-Earle's conditions did not result in an increased intracellular Calcium2+concentration (Figure 5(A)). Similarly, the withdrawal of halothane from a Potassium+-Earle's medium illustrated in Figure 5(B) failed to initiate a detectable transient Calcium2+rise, as was observed with the same cells when bathed in a normal Earle's solution (see also Figure 2). An absence of halothane-induced Calcium (2+)-decrease and/or cytosolic Calcium2+rise after halothane removal was confirmed in five additional experiments carried out on TG-treated cells superfused with a Potassium+-Earle's medium. These observations provide strong evidence that a depolarization of the cell potential is sufficient to impair the effects of halothane on internal Calcium2+, suggesting that most of the inhibitory action of halothane involves a depolarization of the cell potential. Because cells bathed in a Potassium+-Earle's medium are maintained in a depolarized state, there should be a minimal impact on the cell potential due to an inhibition by halothane of the Potassium(Calcium2+) conductance, thereby explaining the lack of halothane effects on Calcium2+influx, as observed. In addition, it is clear from the fura-2 measurements illustrated in Figure 5(A and B) that reperfusion with a normal Earle's solution after exposure to a Potassium+-Earle's medium causes an important increase of the cytosolic Calcium2+concentration. This latter result would be in agreement with the expected voltage dependency of the capacitative Calcium2+influx triggered either by TG or bradykinin, in which more negative membrane potential values have been associated with an enhanced Calcium2+entry. .
The variation in membrane potential of bradykinin-stimulated BAE cells in response to halothane application is illustrated in Figure 6. The bradykinin-evoked hyperpolarization shown in Figure 6(A) is in agreement with the potential measurements reported by Mehrke and others in whole-cell patch-clamp experiments carried out on cultured BAE cells. [17,33]More importantly, Figure 6(A) provides direct evidence of a halothane-evoked depolarization with a magnitude comparable to the hyperpolarization triggered by bradykinin. These measurements offer additional support to the conclusions drawn from the cell-attached experiments illustrated in Figure 6(B), where the amplitude of the current jumps resulting from the openings of the Calcium2+-independent IK1channel was used to monitor membrane potential changes. It is clear, from these experiments, that part of the halothane action on the Calcium2+response of BAE cells to bradykinin involves a depolarization of the cell potential, which is expected, in turn, to contribute to the Calcium2+influx inhibition observed in halothane conditions. In addition, Figure 6(B) shows that there is a correlation between the presence of halothane in the external medium, a depolarization of the cell potential, and a reduced activity of the Potassium(Calcium2+) channels present in these cells. These results suggest that the halothane-induced depolarization reported in Figure 6(A) is related to an inhibition of the Potassium(Calcium2+) channels under these conditions.
A direct inhibitory action of halothane on the Potassium(Calcium2+) channel in BAE cells was observed in a series of inside-out patch-clamp experiments (Figure 8). Halothane was reported to decrease the activity of a large variety of potassium selective channels, including several Calcium2+-activated potassium channels. [42–46]The halothane concentration needed for half inhibition of the Potassium(Calcium2+) channel of large conductance in smooth muscle cells and for the intermediate conductance Potassium(Calcium2+) channel of red blood cells was evaluated at 0.5 mM, a value in agreement with the results presented in Figure 8. The inside-out measurements presented in Figure 8, therefore, are consistent with the whole cell recordings in Figure 6(A), which show a near complete inhibition by halothane of the bradykinin-evoked hyperpolarization. However, the effect of halothane and isoflurane on the activity of the Potassium(Calcium2+) channels in stimulated BAE cells may not exclusively involve a direct inhibition of the Potassium(Calcium2+) channel gating processes, as illustrated in Figure 8, but may also include an indirect effect coming from a decreased intracellular Calcium (2+) concentration.
The fura-2 + cell-attached recordings in Figure 7provide direct evidence for a correlation between the presence of halothane in the external medium, an absence of Potassium(Calcium2+) channel activity, and a low cytosolic Calcium2+concentration. Together, these observations are compatible with a positive feedback mechanism in which volatile anesthetics inhibit the Potassium(Calcium2+) channels in BAE cells directly, thereby causing a depolarization of the cell potential, a reduced Calcium2+influx, and a lowering of the internal Calcium2+concentration (Figure 1(C, D), Figure 2, and Figure 7). This latter effect should, in turn, fuel the depolarization process by enhancing the inhibition of the Potassium(Calcium2+) channel activity. However, despite the importance of this mechanism, we cannot totally rule out a direct interaction of the anesthetics, with the structures responsible for the agonist-evoked Calcium2+entry. For instance, isoflurane was found to initiate a clear decrease in Calcium2+concentration in bradykinin-stimulated cells (Figure 1(D)), without totally inhibiting Potassium(Calcium2+) channel activity (Figure 8). These observations suggest either that a total inhibition of the Potassium(Calcium2+) channel activity may not be required to initiate a cell depolarization of sufficient magnitude to impair the bradykinin-evoked Calcium2+influx, or that part of the effect of halothane and isoflurane on Calcium2+influx involves a direct inhibition of the Calcium2+entry mechanism. In fact, there is increasing evidence that volatile anesthetics, such as halothane, bind competitively to specific hydrophobic regions of proteins. Because halothane has been reported to interact with a large variety of ionic channels, this anesthetic is likely to affect Calcium2+permeable channels, such as the second messenger-operated Calcium2+channel described by Luckhoff and Clapham in BAE cells or the Calcium2+permeable channel activated by Calcium2+store depletion reported by Vaca and Kunze using the same cell preparation. Such inhibitory effects would contribute to further decreasing the capacitative Calcium (2+) influx, leading to a more significant reduction, by halothane or isoflurane, of the cell internal Calcium2+concentration. The significance of this mechanism for BAE cells remains to be established, because no additional effect of halothane accountable by a direct interaction with the Calcium2+entry machinery was observed in cells maintained in a depolarized state (Figure 5).
The cardiovascular effects of halothane and isoflurane are likely to be manifold. Our results show that halothane and isoflurane decrease the Calcium2+availability in endothelial cells mainly through an action on the agonist-evoked Calcium2+entry. This phenomenon involved an effect on the cell membrane potential characterized by a cell depolarization consequent to an inhibition by the volatile anesthetics of the Potassium(Calcium2+) channels present in these cells. The resulting decrease in Calcium2+availability is likely to interfere with NO production by modulating the NO synthase activity. However, the overall contribution of this mechanism to the inhibition by halothane and isoflurane of the endothelium-dependent vascular smooth muscle relaxation process needs further investigation. Other mechanisms, such as a direct effect of halothane on NO stability, were shown to play a significant role in this regard. Similarly, Johns et al. provided evidence that halothane could affect the activity of the NO synthase independently of cytosolic Calcium2+variations. An action of halothane and isoflurane on internal Calcium2+homeostasis would, nevertheless, constitute a general phenomenon that would affect not only NO production but also the entire Calcium2+signalling process in these cells.
The authors thank Dr. M. J. Coady, who kindly read the revised version of the manuscript, and M. P. Drouilly, for technical assistance in the fura-2 measurements with isoflurane.