Free
Pain Medicine  |   June 2002
Isoflurane-induced Dilation of Porcine Coronary Microvessels Is Endothelium Dependent and Inhibited by Glibenclamide
Author Affiliations & Notes
  • A. Kurt Gamperl, Ph.D.
    *
  • Travis W. Hein, Ph.D.
  • Lih Kuo, Ph.D.
  • Brian A. Cason, M.D.
    §
  • *Assistant Professor of Biology, Portland State University, Portland, Oregon; current position: Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. †Research Assistant Professor, ‡Professor, Cardiovascular Research Institute, Department of Medical Physiology, Texas A&M University System Health Science Center, College Station, Texas. §Professor and Vice Chairman, Department of Anesthesia and Perioperative Care, University of California–San Francisco, and Chief, Anesthesiology Service, Veterans Affairs Medical Center.
  • Received from the Department of Anesthesia and Perioperative Care, University of California–San Francisco, and the Anesthesiology Service, Veterans Affairs Medical Center, San Francisco, California.
Article Information
Pain Medicine
Pain Medicine   |   June 2002
Isoflurane-induced Dilation of Porcine Coronary Microvessels Is Endothelium Dependent and Inhibited by Glibenclamide
Anesthesiology 6 2002, Vol.96, 1465-1471. doi:
Anesthesiology 6 2002, Vol.96, 1465-1471. doi:
ISOFLURANE has marked effects on blood flow and vascular resistance in many tissues, including the heart. 1–5 However, the primary mechanisms responsible for the direct effect of isoflurane on coronary vascular resistance, and indeed the nature of the vasomotor response of coronary microvessels to this volatile anesthetic, are still in question. In vivo  and in situ  studies that examined the effects of isoflurane on coronary perfusion indicate that isoflurane increases coronary blood flow 4-11 and dilates epicardial coronary vessels (20–450-μm ID), 12 strongly implying that resistance in the smallest coronary arterioles must therefore be decreased. Based on these data, isoflurane is widely regarded as a coronary vasodilator. Despite this evidence, several in vitro  studies of cannulated microvessels 13–16 report that isoflurane is a potent vasoconstrictor of coronary resistance arterioles. These results are inconsistent with the preponderance of data from the aforementioned in vivo  studies as well as a recent in vitro  coronary microvessel study (Zhou et al.  17), and have lead to the publication of several editorials and letters of correspondence. 18–20 
Because the response of coronary resistance arterioles to isoflurane is paramount to our understanding of how this volatile anesthetic affects myocardial blood flow, it is imperative to clarify whether isoflurane directly constricts or dilates these vessels. In this study we used small swine coronary arterioles (75 ± 4.0 μm) that developed spontaneous (intrinsic) tone and were not subject to preconstriction or predilation, to examine the dose-dependent effect of clinically relevant concentrations of isoflurane on coronary microvessel diameter. In addition, to address the question of mechanisms of isoflurane-induced vasodilation in these vessels, we: (1) measured the dose–response of coronary arterioles to isoflurane in the presence and absence of glibenclamide, a blocker of adenosine triphosphate–sensitive potassium (KATP) channels, and iberiotoxin, a blocker of Ca2+-activated potassium (KCa) channels; and (2) examined the role of the endothelium in the pharmacologic response to isoflurane by measuring vasomotor responses in vessels before and after endothelial denudation. This study clearly demonstrates that isoflurane-mediated dilation of coronary microvessels is not dependent on pharmacologic preconstriction and provides further evidence that dilation is the predominant response of coronary arterioles to isoflurane.
Materials and Methods
General Preparation
This experimental protocol was approved by the Laboratory Animal Care Committee at Texas A&M University (College Station, TX) and followed the guidelines for animal use published by the American Physiologic Society. Pigs (n = 22) of either sex that weighed 7–11 kg were sedated with an intramuscular injection of tiletamine and zolazepam (1:1, 4.4 mg/kg) and xylazine (2.2 mg/kg), anesthetized with pentobarbital (20 mg/kg), and injected with heparin (1,000 U/kg) via  a marginal ear vein. After tracheotomy and intubation, the animals were ventilated with room air. A left thoracotomy was performed, and the heart was electrically fibrillated, excised, and placed in cold (5°C) saline.
Isolation and Cannulation of Microvessels
The techniques for identification and isolation of porcine coronary microvessels were described previously. 21 In brief, a mixture of india ink and gelatin in physiologic salt solution (PSS) containing 145.0 mm NaCl, 4.7 mm KCl, 2.0 mm CaCl2, 1.17 mm MgSO4, 1.2 mm NaH2PO4, 5.0 mm glucose, 2.0 mm pyruvate, 0.02 mm EDTA, and 3.0 mm MOPS was perfused into the left anterior descending artery (0.3 ml) and the circumflex artery (0.4 ml) to enable the visualization of coronary microvessels. Subepicardial arteriolar vessels (40–108-μm ID and 0.6–1.0 mm long without branches) from the left anterior descending or circumflex arteries were selected and carefully dissected from the surrounding cardiac tissue under cold (5°C) PSS containing bovine serum albumin (1%; Amersham, Arlington Heights, IL) at pH 7.4. Each isolated arteriole was then transferred for cannulation to a Lucite vessel chamber (2 ml in volume) containing PSS–albumin solution equilibrated with room air at ambient temperature. One end of the microvessel was cannulated with a glass micropipette (40 μm in tip diameter) filled with filtered PSS–albumin solution, and the microvessel was securely tied to the pipette with 11-O ophthalmic suture (Alcon, Forth Worth, TX). The ink–gelatin solution inside the vessel was flushed out at low perfusion pressure (< 15 mmHg), and the other end of the vessel was cannulated with a second micropipette and tied with a suture. We previously showed that the ink–gelatin solution has no detrimental effect on either endothelial or vascular smooth muscle function. 22,23 
Instrumentation
After microvessel cannulation, the chamber was transferred to the stage of an inverted microscope (model IM35; Zeiss, Thornwood, NY) coupled to a CCD camera (KP-161; Hitachi, Tokyo, Japan) and a videomicrometer (Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX). Vessel ID was measured with a precision of ± 1 μm throughout the experiment using videomicroscopy (160× final magnification) and a calibrated video caliper (795 × 596 pixel resolution). The micropipettes were connected to independent reservoir systems, and intraluminal pressures were measured through sidearms of the two reservoir lines by low-volume displacement strain-gauge transducers (Statham P23 Db; Gould, Cleveland, OH). The isolated vessels were pressurized without flow by setting both reservoirs at the same hydrostatic level. Leaks were detected by differences between reservoir pressure and intraluminal pressure. Preparations with leaks were excluded from further study.
Experimental Protocols
The cannulated arterioles were bathed in PSS–albumin solution (pH 7.4 at 37°C) equilibrated with room air and maintained at 36–37°C by connecting the chamber's water jacket to an external heat exchanger. The microvessels were set to their in situ  length and allowed to develop basal tone (< 70–75% of maximal ID) at 45 mmHg intraluminal pressure without flow. These pressures approximate the estimated intraluminal pressures for microvessels of this size in vivo  . 24 After the vessels developed basal tone (30–40 min), the direct effect of isoflurane on microvascular tone was assessed by exposing each arteriole to increasing concentra-tions of isoflurane (0.16, 0.29, 0.43, 0.57, and 0.75 mm; 1 minimum alveolar concentration [MAC]= 0.3 mm). Isoflurane concentrations were achieved by sequentially injecting small quantities (20–45 μl) of isoflurane-saturated PSS–albumin solution into the well using a Hamilton syringe and mixing the isoflurane within the well by repeatedly (three to four times) drawing buffer into the Hamilton syringe and slowly reinjecting it. Measurements of vessel ID were taken approximately 1 min after each isoflurane injection was mixed within the well, and injections of isoflurane were separated by approximately 2 min. Vessel ID was very stable between 1 and 2 min, and changes in coronary vessel ID were never observed after single or repeated injections of vehicle (PSS–albumin) into the well.
The possible involvement of vascular smooth muscle KATPand KCachannels in isoflurane-induced vasodilation was examined by extraluminal incubation (30 min) of intact vessels with the specific inhibitors glibenclamide (1 μm) and iberiotoxin (100 nm), respectively. 25,26 The specificity of glibenclamide as a KATPchannel inhibitor was verified by testing its ability to block vasodilation in response to pinacidil (106m, a specific KATPchannel opener 27), and the efficacy of iberiotoxin was confirmed by inhibition of arachidonic-induced (10 μm) coronary arteriolar dilation. 26 In addition, to evaluate whether glibenclamide had a nonspecific effect on vasodilatory function, the dose-dependent dilation of isolated vessels to sodium nitroprusside (SNP; 109to 105m) was examined in the absence and presence of extraluminal glibenclamide (1 μm for 30 min).
To assess the role of the endothelium in the vascular response to isoflurane, the diameter–isoflurane relation was studied before and after endothelial denudation. To remove the endothelial cells, a nonionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 0.4%), was intraluminally perfused into the vessel for 1–2 min. 28 After removal of the endothelium, as verified by the absence of vasodilation to the endothelium-dependent vasodilator bradykinin (1 nm), the vessel was perfused with PSS–albumin for 5 min to remove the CHAPS. To ensure that vascular smooth muscle function was not compromised by CHAPS treatment, dose-dependent dilation in response to SNP (109to 105m) was also examined in vessels before and after denudation.
At the end of each experiment, vessels were relaxed completely with SNP (100 μm) to obtain the maximal diameter at 45 mmHg intraluminal pressure.
Isoflurane Measurements
The reported cumulative isoflurane concentrations were determined in separate experiments using gas chromatography. Briefly, after the injection of each small volume of isoflurane-saturated buffer and complete mixing within the well, the PSS–albumin solution within the well (approximately 2 ml) was drawn into a preweighed, volume-calibrated, gas-tight syringe. A volume of air was drawn into the syringe to achieve a total volume of 20 ml, and the air–PSS solution was equilibrated in a 37°C rotary bath for 2 h. After equilibration, a gas sample was injected into a precalibrated gas chromatograph.
In preliminary experiments using this albumin-containing MOPS buffer, we determined the buffer:gas partition coefficient for isoflurane at 37°C to be 0.80 ± 0.01, with a coefficient of variation of 1.25. The concentration of isoflurane in the PSS–albumin solution was calculated as:EQUATION
Chemicals
Drugs were obtained from Sigma Chemical Co. (St. Louis, MO), except as specifically stated. Isoflurane was obtained as Forane (Ohmeda, Liberty Corner, NJ). Bradykinin and SNP were dissolved in PSS. Glibenclamide was dissolved in dimethyl sulfoxide, and pinacidil and iberiotoxin were dissolved in ethanol as stock solutions (10 mm). Subsequent concentrations of these drugs were diluted in PSS. The final concentrations of dimethyl sulfoxide and ethanol in the vessel bath were 0.03 and 0.1%, respectively. Vehicle control studies indicated that these final concentrations of dimethyl sulfoxide and ethanol had no effect on arteriolar function.
Data Analysis
In this study, only vessels that met the criteria for reactive isolated arterioles were included for data analysis. These criteria were as follows: (1) the development of spontaneous tone (i.e.  , contraction to < 75% of maximal diameter at 37°C); (2) myogenic responsiveness when the 37°C PSS–albumin solution in the vessel chamber was replaced with fresh room-temperature solution; and (3) nonsignificant differences in dose-dependent vasodilation to SNP (109to 105m) before and after experimental treatment. 26 
Vasodilatory responses to isoflurane, in the absence and presence of K+channel blockers and after endothelial removal, were analyzed by normalizing changes in arteriolar diameter to the dilation observed for each vessel after exposure to 104m SNP, which induced maximal vasodilation. In this formula, resting diameter refers to vessel diameter after the development of spontaneous tone.
Statistics
Statistical comparisons between groups (isoflurane vs.  isoflurane + glyburide, isoflurane vs.  isoflurane + iberiotoxin, and isoflurane before and after denudation) were made at each isoflurane concentration using paired student t  tests. Repeated-measures analysis of variance, followed by multiple contrasts, were used to determine the concentrations of isoflurane and SNP that caused significant vasodilation. Student t  tests were also used to examine whether glibenclamide or denudation reduced the vasodilatory response of isolated arterioles to SNP. All statistical analyses were performed using Statview and SuperAnova statistical software (Abacus Concepts, Berkeley, CA). Differences were considered significant at P  < 0.05. Values presented in figures and throughout the text are mean ± SEM.
Results
The isolated coronary arterioles (n = 22) had a resting ID of 75 ± 4 μm (range, 41–108 μm) 30–40 min after being pressurized to 45 mmHg. Thus, the basal tone developed by these vessels was 69 ± 2% of maximal diameter (109 ± 5 μm). Isoflurane caused significant dose-dependent vasodilation in all of the control (untreated) coronary microvessels tested (fig. 1). This vasodilation was 6.0 ± 0.7 μm at an isoflurane concentration of 0.16 mm (approximately 0.5 MAC) and 25.3 ± 2.1 μm at the highest concentration of isoflurane tested (0.75 mm; approximately 2.5 MAC). These values represent 18.1 ± 1.7% and 74.1 ± 3.3%, respectively, of that observed with 104SNP (34 ± 3 μm).
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 104m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 104m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
×
Incubation of microvessels with the KCablocker iberiotoxin (0.1 μm) did not affect isoflurane-mediated vasodilation. Coronary arteriole ID was significantly increased at all isoflurane concentrations, and there was no difference in the degree of dilation between untreated and iberiotoxin-exposed vessels at any isoflurane concentration (fig. 2). The failure of iberiotoxin to influence the isoflurane dose–response curve was not caused by ineffective KCablockade. Coronary dilation in response to arachidonic acid (10 μm) was reduced from 75 ± 2% to 8 ± 2% of maximal dilation after iberiotoxin treatment.
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P  < 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P  < 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
×
In contrast, blockade of KATPchannels with glibenclamide (1.0 μm) greatly diminished the degree of vasodilation in response to isoflurane (fig. 2). Isoflurane failed to increase microvessel ID at both 0.15 and 0.29 mm. In addition, glibenclamide exposure caused a downward displacement of the isoflurane dose–response curve. KATPchannel blockade significantly (P  < 0.05) reduced isoflurane-mediated dilation as compared with control vessels at 0.43 and 0.59 mm, and the decreases observed at 0.29 and 0.75 mm just failed to reach significance (P  = 0.06 for both). These reductions in isoflurane-mediated dilation were not likely the result of the indirect affects of glibenclamide on smooth muscle function. Glibenclamide almost completely blocked pinacidil-mediated vasodilation (control: 75 ± 4%, glibenclamide: 11 ± 5% of maximal dilation, n = 6), and glibenclamide pretreatment (1.0 μm) failed to affect the dose response of coronary microvessels to SNP (fig. 3).
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
×
Bradykinin-induced vasodilation was completely eliminated (control: 85 ± 4%vs.  denudation: 3 ± 1% of maximal dilation, n = 6) after CHAPS treatment, confirming that the endothelium of the vessel had been removed. Endothelial removal significantly reduced the capacity of isoflurane to dilate coronary microvessels (fig. 4). Isoflurane only increased microvessel ID sig-nificantly at the two highest concentrations (0.59 and 0.75 mm). In addition, with the exception of 0.29 mm isoflurane, the degree of isoflurane-mediated vasodilation was significantly reduced after vessel denudation. For example, denudation reduced the amount of dilation in response to 0.75 mm isoflurane by 70%. Again, the observed decrease in isoflurane-mediated vasodilation was not the result of indirect effects on smooth muscle function. Endothelium-denuded microvessels had similar dose-dependent responses to SNP as vessels with an intact endothelium (fig. 3).
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
×
Discussion
Dose-related Coronary Arteriolar Vasodilation
The most important findings of our study are that isoflurane caused significant dose-related vasodilation (6–25 μm) in isolated coronary resistance arterioles, and that this vasodilation was near-maximal at the highest dose of isoflurane (0.75 mm; approximately 2.5 MAC). These results are in agreement with the findings of Zhou et al.  17 and numerous in vivo  studies, 4–7,9–12 and add significantly to the weight of evidence which indicates that the predominant effect of isoflurane on the coronary vasculature is vasodilation.
Why our results contrast with the studies of Park et al.  , 13–16 which report isoflurane-mediated coronary vasoconstriction, is unknown. However, it is apparent that neither differences in the species studied or in vessel size can account for the discrepancy in results. In the current study, we used swine coronary arterioles with an ID of 75 ± 4 μm (range, 41–108 μm). These microvessels were smaller than the arterioles studied by Park et al.  in rabbits (139 ± 34 μm), 13 but similar to the size of vessels studied by that group in rats (99 ± 15 μm) 15 and swine (range, 60–150 μm). 16 Furthermore, our coronary arterioles were clearly “resistance” vessels, because the greatest fraction of the resistance in the coronary circulation of arrested swine hearts resides in arterial vessels less than 100 μm. 29 
There is no simple mechanistic explanation as to why isoflurane caused vasoconstriction of in vitro  vessels in the studies by Park et al.  , 13–16 but vasodilation in this study and those of Zhou et al.  17 and Park et al.  30 However, methodologic differences between studies cannot be ruled out as a potential contributor to these conflicting findings. One potentially important methodologic difference between the current study and the studies of Park et al.  13–16 was the criteria used as the primary indicator of the success of the arterial preparations, and thus the physiologic state of the arterioles. In the studies by Park et al.  , 13–16 microvessel viability was determined by exposing microvessels to potassium chloride (maximum concentration, 100 mEq/l) at the end of the experiments and by ensuring that vessels constricted by at least 10%. 13 In contrast, we used three criteria to establish viability of coronary microvessels: the development of intrinsic tone within 30 min of being stretched to in situ  length, clear myogenic responsiveness to a 10–12°C decrease in bath temperature, and consistent and significant dose-dependent vasodilation in response to SNP before and after treatments. We do not suggest that only isolated vessels with intrinsic tone should be studied. However, the development of intrinsic tone is an excellent indicator of the relative integrity of the physiologic systems of the microvessel, and results obtained with such vessels are most likely to represent those in vivo  .
Other methodologic differences between this study and those by Park et al.  13–16 include oxygen concentrations in the bathing solution and the method of anesthetic delivery. In the studies by Park et al.  , the vessels were continuously bathed in a Kreb's buffer solution gassed with 95% O2–5% CO2(oxygen tension > 400 mmHg), and isoflurane was added to the buffer solution using an in-line vaporizer. In the current study, the PSS–albumin solution was equilibrated with room air, and isoflurane concentrations were achieved by sequentially injecting small quantities (20–45 μl) of isoflurane-saturated PSS–albumin solution into the well. However, it is unlikely that either of these factors can explain the contradictory results obtained in the two studies. Park et al.  14 demonstrated that vasoconstrictive responses to isoflurane were similar in microvessels bathed in hyperoxic (95% O2–5% CO2) and nonhyperoxic buffer. In addition, Zhou et al.  17 demonstrated that, although abrupt versus  gradual administration of isoflurane via  a vaporizer caused differences in the magnitude of in vitro  coronary arteriolar responses, isoflurane always produced significant vasodilation.
Isoflurane-induced Vasodilation Inhibited by Glibenclamide
Isoflurane-induced coronary vasodilation in this model was inhibited by glibenclamide, a blocker of KATPchannels, but not by iberiotoxin, an inhibitor of KCachannels. This supports the idea that isoflurane-induced vasodilation of resistance coronary arterioles (< 100 μm) is mediated through cellular hyperpolarization caused by the opening of KATPchannels. These findings agree with and support the in vitro  study of Zhou et al.  , 17 which looked at medium-sized coronary arterioles (172 ± 51 μm), and confirm previous in vivo  work from our laboratory 6 and from Crystal et al.  8 that showed glibenclamide diminished isoflurane-mediated increases in coronary blood flow. Whether the KATPchannels that participate in the vasodilatory response of coronary microvessels to isoflurane are located in coronary vascular smooth muscle or the endothelium is still unknown. Neither this study or that of Zhou et al.  17 performed glibenclamide experiments on denuded vessels, and the opening of endothelial KATPchannels can reduce vascular smooth muscle tone by either the production of second messengers or by the direct transduction of electrical potentials. KCachannels, on the other hand, do not appear to be involved in isoflurane-induced coronary vasodilation. This result is concordant with the findings of Larach and Schuler, 31 who showed that TEA+(a blocker of KCa) did not significantly reduce halothane-mediated vasodilation in isolated rat hearts.
Role of the Endothelium in Isoflurane-induced Coronary Vasodilation
Isoflurane-induced coronary vasodilation was inhibited by approximately 70% after endothelial denudation in these experiments, but dose-dependent and maximal vasodilations in response to the endothelium-independent vasodilator nitroprusside were not affected. These findings provide the first evidence that isoflurane-induced vasodilation of coronary microvessels (< 100 μm) is largely endothelium dependent. However, at present, no mechanism(s) has been clearly identified that can explain this result. Crystal et al.  , 32 who used a dog model of isolated coronary perfusion, found that the nitric oxide synthase inhibitor N  -nitro-l-arginine methylester did not reduce isoflurane-induced coronary vasodilation even though it significantly reduced the endothelium-dependent vasodilation caused by acetylcholine. Although species differences must be considered (dogs in the study by Crystal et al.  vs.  swine in the current study), these data suggest that the endothelium-dependent vasodilation measured in this study was not the result of enhanced nitric oxide production. Lischke et al.  33 showed that bradykinin-stimulated dilation of the rat coronary circulation after nitric oxide–prostaglandin GI2blockade was significantly attenuated by isoflurane (2 vol%). This attenuation suggests that isoflurane impairs the cytochrome P450-dependent synthesis of endothelium-derived hyperpolarizing factor in the coronary microcirculation. In addition, there are conflicting reports about the role of prostanoids in isoflurane-mediated vascular dilation. Stone and Johns 34 demonstrated that isoflurane dilates rat aortic rings at higher concentrations and suggested that this vasodilatory effect involved the release of a prostanoid from the endothelium. In contrast, Loeb et al.  35 showed that halothane inhibits bradykinin-stimulated prostacyclin production in cultured bovine aortic endothelial cells.
Methodologic Critique and Limitations of the Current Study
The most important limitation of the current study was that it was performed in vitro  , using vessels in a no-flow state. The pharmacologic effects of isoflurane on in vivo  vessels may be different and more complex, particularly because of the potential contribution and amplifying effect of flow-mediated vasodilation. For example, evidence suggests that volatile anesthetics, including isoflurane, inhibit flow-mediated coronary vasodilation. 36 Furthermore, coronary arterioles in vivo  are surrounded by myocardium, and coronary blood flow is normally regulated so that it matches myocardial oxygen demand. Isoflurane, by altering metabolic demands of myocardial tissue, may cause indirect changes in coronary blood flow (i.e.  , metabolically mediated vasoconstriction) that partially offset isoflurane-induced vasodilation. Although experiments such as the current one facilitate the study of direct pharmacologic effects, extrapolation of these results to the intact, blood-perfused organ must be made with caution.
Another potential drawback of this model is that the main pharmacologic agent to be tested, isoflurane, was applied extraluminally. Vessel adventitia and smooth muscle would thus be more directly exposed to isoflurane than would the vessel endothelium. However, the vessels tested are extremely small, and the fact that extraluminal administration mimics the physiologic effects of intraluminal administration (via  the bloodstream) 6,8 suggests that this was not a limiting factor.
In conclusion, using swine coronary resistance arterioles with innate tone, we found that isoflurane caused dose-dependent vasodilation, and this marked vasodilation was inhibited by the KATPchannel blocker glibenclamide and was predominantly endothelium-dependent. Our finding that isoflurane dilates coronary microvessels agrees with numerous in vivo  studies which show that isoflurane reduces coronary vascular resistance and uncouples myocardial oxygen supply from demand, and provides further experimental evidence that vasodilation, not vasoconstriction, is the predominant action of isoflurane in the coronary circulation.
References
Fujiwara Y, Murray PA: Effects of isoflurane anesthesia on pulmonary vascular responses to K+ATPchannel activation and circulatory hypotension in chronically instrumented dogs. A nesthesiology 1999; 90: 799–811Fujiwara, Y Murray, PA
Iida H, Ohata H, Iida M, Watanabe Y, Dohi S: Isoflurane and sevoflurane induce vasodilation of cerebral vessels via ATP-sensitive K+channel activation. A nesthesiology 1998; 89: 954–60Iida, H Ohata, H Iida, M Watanabe, Y Dohi, S
Sundeman H, Biber B, Raner C, Winsö O: Autoregulation and vasodilator responses by isoflurane and desflurane in the feline renal vascular bed. Acta Anaesthesiol Scand 1997; 41: 1180–6Sundeman, H Biber, B Raner, C Winsö, O
Cason BA, Verrier ED, London MJ, Mangano DT, Hickey RF: Effects of isoflurane and halothane on coronary vascular resistance and collateral myocardial blood flow: Their capacity to induce coronary steal. A nesthesiology 1987; 67: 665–75Cason, BA Verrier, ED London, MJ Mangano, DT Hickey, RF
Crystal GJ, Kim S-J, Czinn EA, Salem MR, Mason WR, Abdel-Latif M: Intracoronary isoflurane causes marked vasodilation in canine hearts. A nesthesiology 1991; 74: 757–65Crystal, GJ Kim, S-J Czinn, EA Salem, MR Mason, WR Abdel-Latif, M
Cason BA, Shubayev I, Hickey RF: Blockade of adenosine triphosphate-sensitive potassium channels eliminates isoflurane-induced coronary artery vasodilation. A nesthesiology 1994; 81: 1245–55Cason, BA Shubayev, I Hickey, RF
Crystal GJ, Czinn EA, Silver JM, Salem MR: Coronary vasodilation by isoflurane: Abrupt versus gradual administration. A nesthesiology 1995; 82: 542–9Crystal, GJ Czinn, EA Silver, JM Salem, MR
Crystal GJ, Gurevicius J, Salem MR, Zhou X: Role of adenosine triphosphate–sensitive potassium channels in coronary vasodilation by halothane, isoflurane, and enflurane. A nesthesiology 1997; 86: 448–58Crystal, GJ Gurevicius, J Salem, MR Zhou, X
Buffington CW, Romson JL, Levine A, Duttlinger NC, Huang AH: Isoflurane induces coronary steal in a canine model of chronic coronary occlusion. A nesthesiology 1987; 66: 280–92Buffington, CW Romson, JL Levine, A Duttlinger, NC Huang, AH
Hickey RF, Sybert PE, Verrier ED, Cason BA: Effects of halothane, enflurane, and isoflurane on coronary blood flow autoregulation and coronary vascular reserve in the canine heart. A nesthesiology 1988; 68: 21–30Hickey, RF Sybert, PE Verrier, ED Cason, BA
Hickey RF, Cason BA, Shubayev I: Regional vasodilating properties of isoflurane in normal swine myocardium. A nesthesiology 1994; 80: 574–81Hickey, RF Cason, BA Shubayev, I
Conzen PF, Habazettl H, Vollmar B, Christ M, Baier H, Peter K: Coronary microcirculation during halothane, enflurane, isoflurane, and adenosine in dogs. A nesthesiology 1992; 76: 261–70Conzen, PF Habazettl, H Vollmar, B Christ, M Baier, H Peter, K
Park KW, Dai HB, Lowenstein E, Darvish A, Sellke FW: Heterogeneous vasomotor responses of rabbit coronary microvessels to isoflurane. A nesthesiology 1994; 81: 1190–7Park, KW Dai, HB Lowenstein, E Darvish, A Sellke, FW
Park KW, Dai HB, Lowenstein E, Darvish A, Sellke FW: Oxygen-derived free radicals mediate isoflurane-induced vasoconstriction of rabbit coronary resistance arteries. Anesth Analg 1995; 80: 1163–7Park, KW Dai, HB Lowenstein, E Darvish, A Sellke, FW
Park KW, Dai HB, Lowenstein E, Sellke FW: Vasomotor responses of rat coronary arteries to isoflurane and halothane depend on preexposure tone and vessel size. A nesthesiology 1995; 83: 1323–30Park, KW Dai, HB Lowenstein, E Sellke, FW
Park KW, Lowenstein E, Dai HB, Lopez JJ, Stamler A, Simons M, Sellke FW: Direct vasomotor effects of isoflurane in subepicardial resistance vessels from collateral-dependent and normal coronary circulation of pigs. A nesthesiology 1996; 85: 584–91Park, KW Lowenstein, E Dai, HB Lopez, JJ Stamler, A Simons, M Sellke, FW
Zhou X, Abboud W, Manabat NC, Salem MR: Isoflurane-induced dilation of porcine coronary arterioles is mediated by ATP-sensitive potassium channels. A nesthesiology 1998; 89: 182–9Zhou, X Abboud, W Manabat, NC Salem, MR
Merin RG, Johns RA: Does isoflurane produce coronary vasoconstriction? A nesthesiology 1994; 81: 1093–6Merin, RG Johns, RA
Crystal GJ: Vasomotor effects of isoflurane in the coronary circulation (letter; comment). A nesthesiology 1996; 84: 1516–8Crystal, GJ
Sellke FW, Park KW, Lowenstein E: Vascular effects of isoflurane: No inconsistency between data (letter). A nesthesiology 1999; 90: 919–20Sellke, FW Park, KW Lowenstein, E
Kuo L, Davis MJ, Chilian WM: Myogenic activity in isolated subepicardial and subendocardial coronary arterioles. Am J Physiol 1988; 255: H1558–62Kuo, L Davis, MJ Chilian, WM
Kuo L, Davis MJ, Chilian WM: Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol 1990; 259: H1063–70Kuo, L Davis, MJ Chilian, WM
Kuo L, Chilian WM, Davis MJ: Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol 1991; 261: H1706–15Kuo, L Chilian, WM Davis, MJ
Chilian WM, Eastham CL, Marcus ML: Microvascular distribution of coronary vascular resistance in beating left ventricle. Am J Physiol 1986; 251: H779–88Chilian, WM Eastham, CL Marcus, ML
Nelson MT: Ca2+-activated potassium channels and ATP-sensitive potassium channels as modulators of vascular tone. Trends Cardiovasc Med 1993; 3: 54–60Nelson, MT
Hein TW, Liao JC, Kuo, L: oxLDL specifically impairs endothelium-dependent, NO-mediated dilation of coronary arterioles. Am J Physiol 2000; 278: H175–83Hein, TW Liao, JC Kuo, L
Nelson MT, Quayle JM: Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995; 268: C799–822Nelson, MT Quayle, JM
Ishizaka H, Kuo L: Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity. Am J Physiol 1997; 273: H104–12Ishizaka, H Kuo, L
Chilian WM: Microvascular pressures and resistances in the left ventricular subepicardium and subendocardium. Circ Res 1991; 69: 561–70Chilian, WM
Park KW, Dai HB, Comunale ME, Gopal A, Sellke FW: Dilation by isoflurane of preconstricted, very small arterioles from human right atrium is mediated in part by K+-ATP channel opening. Anesth Analg 2000; 91: 76–81Park, KW Dai, HB Comunale, ME Gopal, A Sellke, FW
Larach DR, Schuler HG: Potassium channel blockade and halothane vasodilation in conducting and resistance coronary arterioles. J Pharm Exp Ther 1993; 267: 72–81Larach, DR Schuler, HG
Crystal GJ, Kim S-J, Salem MR, Khoury E, Gurevicius J: Nitric oxide does not mediate coronary vasodilation by isoflurane. A nesthesiology 1994; 81: 209–20Crystal, GJ Kim, S-J Salem, MR Khoury, E Gurevicius, J
Lischke V, Busse R, Hecker M: Volatile and intravenous anesthetics selectively attenuate the release of endothelium-derived hyperpolarizing factor elicited by bradykinin in the coronary microcirculation. Naunyn Schmiedebergs Arch Pharmacol 1995; 352: 346–9Lischke, V Busse, R Hecker, M
Stone DJ, Johns RA: Endothelium-dependent effects of halothane, enflurane, and isoflurane on isolated rat aortic vascular rings. A nesthesiology 1989; 71: 126–32Stone, DJ Johns, RA
Loeb AL, O'Brien DK, Longnecker DE: Halothane inhibits bradykinin-stimulated prostacyclin production in endothelial cells. A nesthesiology 1994; 81: 931–8Loeb, AL O'Brien, DK Longnecker, DE
Park KW, Dai HB, Lowenstein E, Sellke FW: Flow-induced dilation of rat coronary microvessels is attenuated by isoflurane but enhanced by halothane. A nesthesiology 1998; 89: 132–42Park, KW Dai, HB Lowenstein, E Sellke, FW
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 104m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
Fig. 1. Increases in porcine coronary arteriolar diameter (n = 22; baseline ID, 75 ± 4 μm) after administration of varying concentrations of isoflurane (0.16–0.75 mm; 0.5–2.5 MAC). Maximal dilation (34 ± 3 μm) was determined by exposing each vessel to 104m sodium nitroprusside. Vertical bars represent 1 ± SEM. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure.
×
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P  < 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
Fig. 2. Effect of potassium-channel blockade on isoflurane-mediated vasodilation in porcine coronary arterioles. Maximal dilation (36 ± 4 μm) was determined by exposing each vessel to 10−4m sodium nitroprusside. Asterisks indicate a significant (P  < 0.05) increase in arteriolar internal diameter as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and glibenclamide-treated vessels at each isoflurane concentration. Baseline values for vessel ID in the control, glibenclamide, and iberiotoxin groups were 77 ± 7, 74 ± 6, and 82 ± 10 μm, respectively. Vertical bars represent 1 ± SEM.
×
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
Fig. 3. Effect of glibenclamide (1 μm) treatment and endothelial removal with the nonionic detergent CHAPS (0.4%) on arteriolar responses to increasing concentrations of sodium nitroprusside. Neither glibenclamide nor endothelial denudation affected the dose response of vessels to sodium nitroprusside. Baseline ID in control vessels was 76 ± 3 μm and was not affected by either treatment. Vertical bars represent 1 ± SEM.
×
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P 
	< 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
Fig. 4. Effect of endothelial denudation (0.4% CHAPS) on isoflurane-mediated vasodilation in porcine coronary microvessels. Asterisks indicate a significant (P  < 0.05) increase in arteriolar ID as compared with vessels before isoflurane exposure. Plus signs indicate a significant difference in isoflurane-mediated vasodilation between control and denuded arterioles at each isoflurane concentration. Vertical bars represent 1 ± SEM. Baseline values for ID in the control and denuded groups were 80 ± 6 and 76 ± 6 μm, respectively.
×