Sevoflurane Selectively Increases Coronary Collateral Blood Flow Independent of K^ATPChannels In Vivo 

All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. All procedures conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (revised, 1996).

Conditioned mongrel dogs were fasted overnight. Anesthesia was induced with intravenous propofol (5 mg/kg). After tracheal intubation, anesthesia was maintained with isoflurane (1.5-2.0%) in 100% oxygen via positive pressure ventilation. A left thoractomy was performed during sterile conditions, and a segment (1.0-1.5 cm) of the left anterior descending coronary artery (LAD) immediately distal to the first diagonal branch was isolated. An ameroid constrictor (Research Instruments and MFG, Corvallis, OR) was placed around the vessel. The diameter of the internal lumen of the constrictor was 2.0-3.0 mm, and its size was chosen for a snug fit around the vessel without producing a stenosis. The chest was closed in layers and pneumothorax evacuated with a chest tube. Each dog received antibiotic (cefazolin, 40 mg/kg; and gentamicin, 4.5 mg/kg) and analgesic (buprenorphine, 0.01 mg/kg) agents in the postoperative period.

Eight weeks after implantation of ameroid constrictors (moderately well developed collaterals), dogs were anesthetized with sodium pentobarbital (15 mg/kg) and sodium barbital (200 mg/kg) and ventilated via positive pressure with oxygen-enriched air (fractional inspired oxygen tension = 0.25) after tracheal intubation. A dual, micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted into the aorta and left ventricle via the left carotid artery to measure arterial and left ventricular (LV) pressures, respectively. Heparin-filled catheters were inserted into the right femoral vein and artery for administration of intravenous fluids and withdrawal of reference arterial blood samples, respectively. The arterial catheter was advanced to the level of the descending thoracic aorta.

A thoracotomy was performed in the left fifth intercostal space, the lung gently retracted, and the heart suspended in a pericardial cradle. A heparin-filled catheter was positioned in the left atrium for injection of radioactive microspheres. A descending thoracic aortic snare was placed to facilitate control of arterial pressure. Segments of the left circumflex coronary artery (LCCA; proximal to the first marginal branch) and LAD (distal to the ameroid constrictor) were dissected free from the surrounding myocardium. A transit time flow probe (Transonic, Ithaca, NY) was positioned around the LCCA to determine coronary blood flow (ml/min), and a fluid-filled catheter was inserted into this vessel for drug infusion. Each dog was anticoagulated with heparin (500 U/kg). A large-bore polyethylene cannula attached to Silastic tubing was inserted in the right carotid artery. The proximal LAD was ligated, a large-bore metal cannula was positioned and secured in the distal arterial segment, and a carotid artery to LAD shunt was established (to ensure patency of the distal LAD perfusion cannula between measurements of retrograde blood flow). Left anterior descending coronary artery perfusion was restored within 3 min after ligation. A segment of tubing perpendicular to the metal cannula was used to measure retrograde coronary flow during interruption of antegrade flow through the carotid artery to LAD shunt (Figure 1). Hemodynamics were monitored continuously on a polygraph and digitized via a computer interfaced with an analog-to-digital converter.

Carbonized plastic microspheres labeled with¹⁴¹Ce,¹⁰³Ru,⁵¹Cr, or⁹⁵Nb were used to measure regional myocardial perfusion as previously described. [13]Microspheres were administered into the left atrium as a bolus injection over 10 s. A timed (2-min) collection of reference arterial flow was started from the femoral arterial catheter a few seconds before the microsphere injection. At the conclusion of each experiment, 10 ml Patent blue dye was injected into the LCCA simultaneously with saline infused intracoronary into the LAD perfusion tubing at equal pressure to delineate the normal and collateral-dependent regions, respectively. Transmural tissue samples were selected from the collateral-dependent (distal to the LAD ameroid constrictor) and normal (LCCA) zones. Tissue samples with a significant degree of overlap flow usually present in the peripheral, lateral, superior, and inferior margins of the ischemic region [14]were not used. Samples were subdivided into subepicardial, midmyocardial, and subendocardial layers of approximately equal thickness and weight. Tissue blood flow (ml [middle dot] min^-1[middle dot] g^-1) was calculated as Q^r[middle dot] C^m[middle dot] C^r-1, where Q^ris the rate of withdrawal of the reference blood flow sample (ml/min); C^mis the activity (cpm/g) of the myocardial tissue sample; and C^ris the activity (cpm) of the reference blood flow sample. Transmural blood flow was considered as the average of subepicardial, midmyocardial, and subendocardial blood flows. Coronary perfusion pressure was determined as the difference between end-diastolic arterial pressure and LV end-diastolic pressure. Large and small LAD collateral conductance and LCCA conductance were calculated as the ratio of LAD retrograde, LAD transmural, and LCCA transmural blood flow, respectively, to coronary perfusion pressure.

Hemodynamics were recorded, radioactive microspheres injected, and LAD retrograde blood flow measured 30 min after completion of the acute surgical preparation. Retrograde blood flow was measured by collecting blood from the LAD cannula into a graduated cylinder for 90 s while the cannula tip was maintained at the level of the left atrium. The initial baseline measurements of retrograde blood flow were repeated (usually three to four times) until consistent volumes were obtained. Thereafter, all measurements were performed in triplicate and the results averaged. Microsphere injections were performed with the retrograde flow cannula open so that retrograde and LAD transmural blood flow were measured simultaneously. [15]This procedure differentiates collateral blood flow into two components. Large vessel coronary collateral blood flow is estimated by retrograde flow measurements, and microvascular collateral flow is determined by measurement of tissue blood flow with radioactive microspheres. [15,16] 

The actions of sevoflurane to increase coronary collateral blood flow were compared with those of the K^ATPchannel agonist, nicorandil, in four groups of dogs. Nicorandil previously has been shown to increase coronary collateral blood flow [12]; however, it is unknown whether nicorandil increases blood flow primarily through large or small collaterals. Experiments were performed in one group of dogs, as a positive control, to characterize the response of the coronary collateral circulation to a known K (^ATP) channel agonist that dilates coronary collaterals. In a second group of experiments, nicorandil was administered after blockade of K^ATPchannels with glyburide. These experiments were performed to characterize the specific role of K^ATPchannel activation as a mechanism through which nicorandil increases coronary collateral blood flow. Although nicorandil also has been shown to possess nitrate-like properties, [17]this K^ATPagonist was chosen as a positive control because the effects of other K^ATPchannel agonists, such as aprikalim, bimakalim, and cromakalim on collateral blood flow, are unknown. The actions of the anesthetic agent sevoflurane on coronary collateral blood flow were evaluated and compared with the findings during K^ATPchannel activation with nicorandil. To determine if K^ATPchannel activation by sevoflurane was responsible for the coronary collateral circulatory effects of this agent, sevoflurane was administered after pretreatment with glyburide in a fourth group of dogs. Finally, nicorandil was administered during sevoflurane to determine if additional coronary collateral vasodilator reserve could be elicited via K (^ATP) channel activation. Nicorandil was administered in a cumulative manner via intracoronary infusions (25, 50, and 100 [micro sign]g/min, 10 min each dose) in four groups of dogs pretreated with intracoronary glyburide (50 [micro sign]g/kg over 10 min) or vehicle (50% polyethylene glycol and 50% ethyl alcohol) in the presence or absence of 1 minimum alveolar concentration (MAC) sevoflurane (2.36% end-tidal). End tidal concentrations of sevoflurane were measured at the tip of the endotracheal tube using an infrared gas analyzer calibrated with known standards before and during experimentation. After administration of glyburide or vehicle, hemodynamics, retrograde flow, LAD small collateral blood flow (microspheres), LCCA tissue blood flow (microspheres), and LCCA blood flow (flow probe) were evaluated and measurements repeated in the presence or absence of sevoflurane (30 min of equilibration) and during a cumulative dose-response to nicorandil. Arterial pressure was maintained at control values by partial thoracic aortic constriction throughout experimentation. Arterial blood gases were maintained within a physiologic range throughout experimentation (pH 7.35-7.41; pCO², 27-35 mmHg; pO², 80-250 mmHg).

Statistical analysis of data within and between groups during baseline conditions and during administration of vehicle, glyburide, sevoflurane, or nicorandil was performed with multiple analysis of variance for repeated measures followed by application of Student's t test with Duncan's correction for multiplicity. Changes within and between groups were considered statistically significant when the probability values was <0.05. All data are expressed as mean +/− SEM.

Fifty-three dogs were instrumented with ameroid constrictors to obtain 36 successful experiments. Six dogs died after implantation of the ameroid constrictors during coronary collateral development. The LAD cannulation was unsuccessful in 11 dogs that survived placement of the ameroid constrictor.

Drug vehicle caused no systemic or coronary hemodynamic effects (Figure 2and Table 1). Administration of nicorandil in vehicle-pretreated dogs (Table 1) significantly (P < 0.05) decreased heart rate during maintenance of constant arterial pressure (aortic snare). Large coronary collateral (retrograde) blood flow and conductance were unchanged (Figure 3). In contrast to findings in large collaterals, nicorandil caused dose-dependent increases in small coronary collateral blood flow (LAD tissue flow measured during diversion of retrograde flow) and conductance (Figure 4). In addition, nicorandil profoundly increased LCCA tissue flow and conductance in a dose-dependent fashion (Figure 5). There were no changes in endo/epi blood flow within or between any experimental groups.

Glyburide caused no systemic or coronary hemodynamic effects (Figure 2and Table 2). In contrast to vehicle-pretreated dogs, administration of nicorandil to glyburide-pretreated dogs (Table 2) did not alter heart rate. Increases in small collateral and LCCA blood flow and conductance (Figure 4and Figure 5) produced by nicorandil were significantly attenuated by glyburide.

Sevoflurane decreased heart rate and increased LV end-diastolic pressure (Table 3) during maintenance of constant arterial pressure in the absence or presence of glyburide (Table 4). Large coronary collateral blood flow (Figure 2) was increased by sevoflurane, but small collateral and LCCA blood flows (Table 3) and conductances (Figure 4and Figure 5) remained unchanged in vehicle-pretreated dogs. After pretreatment with glyburide, sevoflurane increased blood flow through large (Figure 2) and small coronary collaterals and increased large (Figure 3) and small (Figure 4) collateral conductance. Despite K^ATPchannel blockade, increases in collateral blood flow during sevoflurane also were accompanied by unchanged LCCA tissue flow and conductance (Figure 5), demonstrating the selective effect of sevoflurane on coronary collateral blood flow.

In the presence of sevoflurane, nicorandil caused dose-dependent increases in LCCA blood flow and conductance (Figure 5). The highest dose of nicorandil (100 [micro sign]g/min) increased large collateral conductance in the presence but not in the absence of sevoflurane (Figure 3). In addition, low doses of nicorandil increased small collateral blood flow and conductance (Figure 4), but high doses decreased small collateral flow. In the presence of glyburide and sevoflurane, administration of nicorandil caused no further increases in collateral blood flow beyond that caused by sevoflurane alone.

Vasomotor responses remain intact in preexisting [18]or newly developed [19]coronary collateral vessels. Study of the vascular reactivity of coronary collaterals is facilitated by division of collateral blood flow into two separate components. Blood flow through large collaterals is typically measured with retrograde blood flow diverted from the distal segment of an occluded coronary artery. In contrast, blood flow through small coronary collaterals is quantified with radioactive microspheres during diversion of retrograde flow. [18,20]These techniques were used in the current investigation to examine the effects of sevoflurane on the components of coronary collateral blood flow and to determine whether K^ATPchannel activation by this volatile anesthetic agent mediates coronary collateral vasodilation in vivo. The current results demonstrate that sevoflurane selectively increases large and small coronary collateral blood flow independent of K^ATPchannel activation. Sevoflurane had no effect on blood flow to the normal LCCA zone but significantly increased retrograde blood flow (+38%) and small coronary collateral conductance (+70%) in the presence of the K^ATPchannel antagonist, glyburide. Sevoflurane also increased retrograde blood flow in vehicle-pretreated dogs. Because of the variability of collateral flow present at baseline among individual dogs, however, this increase in collateral blood flow was statistically significant only after the data were normalized to baseline flow. Increases in LAD retrograde flow observed during administration of sevoflurane were similar to those previously described during intracoronary administration of nitroglycerin glycerin (+28-38%). [18,21]In contrast, the K^ATPchannel agonist nicorandil alone had no effect on large collateral retrograde flow but caused pronounced increases (+148%) in LCCA normal zone perfusion concomitant with increases (+89%) in small collateral blood flow to the LAD zone.

The current results confirm and extend our previous findings of increased coronary collateral blood flow during sevoflurane-induced anesthesia in a canine model of multivessel coronary artery disease. [5]In contrast to sevoflurane, isoflurane [22]or desflurane [23]did not alter collateral blood flow when arterial pressure was held constant. In another experimental model, desflurane, but not isoflurane or halothane, decreased retrograde coronary collateral blood flow after antegrade embolization of the LAD bed. [24]Therefore, sevoflurane-induced increases in coronary collateral blood flow distinguish this agent from other volatile anesthetic agents. The mechanism by which sevoflurane selectively increases coronary collateral flow is unclear. Activation of K^ATPchannels has been strongly implicated as a major mechanism responsible for coronary vasodilation during isoflurane-, [7,8]halothane-, [6,7]and enflurane-induced [7]anesthesia. The current investigation, however, demonstrates that increases in coronary collateral blood flow by sevoflurane occur independently of K^ATPchannels. Nifedipine has been shown to increase retrograde blood flow to an extent similar to sevoflurane by blockade of voltage-dependent calcium channels. [21]Similar results were obtained with diltiazem and verapamil. [25]Sevoflurane also decreases calcium current in guinea pig myocytes, [26]suggesting that this agent may cause coronary collateral vasodilation by a calcium antagonist-like mechanism. Conversely, other volatile anesthetic agents also inhibit myocardial L-type calcium channels to varying degrees, [27]yet these agents do not increase retrograde blood flow in a similar experimental preparation. Nitroglycerin has been demonstrated to increase retrograde flow through the actions of nitric oxide and cyclic guanosine monophosphate. [21]Whether sevoflurane selectively acts via this signal transduction pathway is unknown. Further study is required to delineate the precise mechanisms responsible for sevoflurane-induced increases in coronary collateral blood flow.

The current results not only demonstrate that sevoflurane selectively increases coronary collateral flow in the presence of glyburide but also that these findings contrast with those obtained with the K^ATPchannel agonist nicorandil. The predominant action of nicorandil was to increase normal zone LCCA flow and conductance. In addition, nicorandil actually decreased blood flow through small collaterals after collateral blood flow was enhanced by sevoflurane. This latter effect most likely occurred because the highest dose of nicorandil decreased collateral perfusion pressure. In the absence of a flow-limiting stenosis in the artery supplying blood to the origin of the collaterals, collateral perfusion pressure is usually assumed to be equal to aortic pressure (Figure 6). A significant reduction in pressure may occur across the proximal LCCA, however, causing a subsequent decrease in perfusion pressure at the origin of the collaterals (Figure 6) during large increases in coronary blood flow produced by K^ATPchannel activation. [28]A decrease in collateral perfusion pressure may reduce blood flow through large and small collaterals and result in an underestimation of their respective conductances. Such a reduction in collateral perfusion pressure during increases in LCCA blood flow probably accounts for the decrease in small collateral blood flow and the apparent decrease in small collateral conductance (Figure 4) that occurred during administration of nicorandil in the presence of sevoflurane. The current results also indicate that blockade of K^ATPchannels may facilitate sevoflurane-induced increases in collateral blood flow. Glyburide may have caused a small degree of regional vasoconstriction that was undetectable by measurements of flow or vascular conductance. A small degree of LCCA arteriolar vasoconstriction could unmask the vasodilator actions of sevoflurane on upstream vascular segments such as large coronary collaterals.

The current findings must be interpreted within the constraints of several possible limitations. The effects of sevoflurane on coronary collateral blood flow may not be solely attributable to direct effects on vascular smooth muscle tone. Decreases in inotropic state and heart rate during sevoflurane may have decreased coronary collateral blood flow through effects to decrease myocardial oxygen consumption. Sevoflurane-induced increases in LV end-diastolic pressure also may have altered phasic intramyocardial collateral blood flow. [29] 

Changes in retrograde flow are thought primarily to reflect vasomotor changes of the epicardial collateral vessels [18]; however, intramural collateral vessels also may contribute a minor component to retrograde flow. [18,30]Although the precise contribution of each component of collateral flow cannot be determined with certainty, and although collateral blood flow derived from the right coronary artery was not measured specifically, the methods used in this investigation allowed quantification of total collateral flow. [18]Increases in retrograde flow and LAD tissue flow may have occurred not only because of changes in collateral conductance but also in response to increases in proximal LCCA conductance. Increases in proximal LCCA conductance would increase collateral perfusion pressure and therefore be expected to increase LCCA tissue flow. Sevoflurane, however, did not increase LCCA blood flow measured with either microspheres or with a transonic blood flow probe. Therefore, it is unlikely that significant proximal LCCA dilation occurred in response to sevoflurane, findings consistent with a previous investigation. [4]Increases in blood flow to collateral-dependent myocardium (LAD tissue flow) could occur because of increases in small collateral conductance or increases in arteriolar conductance in the collateral-dependent bed. It has been suggested, however, that collateral-dependent vasculature is near maximally dilated during retrograde flow diversion, [18]and therefore, increases in LAD tissue flow during sevoflurane-induced anesthesia were more likely mediated by increases in small collateral conductance.

A single intracoronary dose (50 [micro sign]g/kg over 10 min) of glyburide was used in the current experiments. This dose of glyburide alone had no effect on retrograde flow but substantially attenuated the coronary vasodilator effects of the K^ATPchannel agonist nicorandil. Profound decreases in retrograde flow were observed in response to higher doses of glyburide (100-200 [micro sign]g/kg over 10 min) during pilot experiments, confirming previous results indicating that glyburide decreases coronary blood flow in a dose-dependent manner. [8,31]Therefore, the dose of glyburide was chosen specifically to block K^ATPchannels effectively without causing direct coronary vasoconstriction. Additional experiments were performed that confirmed the adequacy of K^ATPchannel blockade with glyburide during nicorandil administration over the time course of these experiments. Nicorandil increased coronary blood flow to 161 +/− 5% of baseline in vehicle-pretreated dogs, whereas glyburide blocked these increases, and coronary blood flow increased to only 117 +/− 11% of baseline. A single end-tidal concentration (1 MAC) of sevoflurane was chosen because lower anesthetic concentrations were not expected to cause detectable changes in collateral blood flow. Higher concentrations of sevoflurane were not examined because maintenance of arterial pressure at baseline values using partial aortic constriction would have been technically difficult and also may have precipitated excessive increases in LV end-diastolic pressure leading to reductions in coronary perfusion pressure. Nicorandil was used as a positive control because this K^ATPchannel agonist previously has been shown to increase coronary collateral blood flow. [12]Nicorandil, however, also may dilate large coronary arteries through nitrate effects independent of the K^ATPchannel. [17]Nonetheless, the doses of nicorandil used in the current investigation produced minimal effects of large coronary collaterals. In contrast, nicorandil dilated the normal arterial vasculature in the LCCA region and small LAD coronary collaterals, actions that were inhibited by glyburide. These findings are consistent with those previously reported, [32]suggesting that nicorandil dilates coronary resistance vessels in normal regions. Finally, coronary conductance was calculated using aortic pressure as an estimate of collateral perfusion pressure. Large and small collateral conductance, however, may have been underestimated during administration of high doses of nicorandil if a reduction in pressure occurred at the origin of the collateral vessels during these experimental conditions.

The current results demonstrate that sevoflurane selectively increases large and small coronary collateral blood flow in the presence of the K^ATPchannel antagonist, glyburide. In contrast, the K^ATPchannel against, nicorandil, caused vasodilation of the normal LCCA perfusion territory that was attenuated by glyburide. The findings suggest that sevoflurane produces coronary collateral vasodilation independent of K^ATPchannels.

The authors thank David Schwabe for technical assistance and Angela Barnes for assistance in preparation of this manuscript.