Isoflurane and the Pulmonary Vascular Pressure-Flow Relation at Baseline and during Sympathetic α- and β-Adrenoreceptor Activation in Chronically Instrumented Dogs 

All surgical procedures and experimental were approved by the institutional Animal Care and Use Committee.

Twelve conditioned mongrel dogs (male, 26 plus/minus 1 kg) were used in this study. All dogs were premedicated with morphine sulfate (10 mg, intramuscular) and were anesthetized with pentobarbital sodium (20 mg/kg, intravenous) and fentanyl citrate (15 micro gram/kg, intravenous). After tracheal intubation, the lungs were mechanically ventilated. Anesthesia was maintained with halothane ([nearly equal] 1.2% end-tidal).

A left thoracotomy was performed via the fifth intercostal space by sterile surgical technique, and the pericardium was incised ventral to the phrenic nerve. Heparin-filled Tygon catheters (1.02 mm ID, Norton, Akron, OH) were inserted into the descending thoracic aorta. left and right atrium, and main pulmonary artery and were secured with purse-string sutures. After careful dissection and isolation, a hydraulic occluder (18 mm ID, Jones, Silver Springs, MD) was loosely positioned around the right main pulmonary artery, and an electromagnetic flow probe (10 mm ID, Zepeda, Seattle, WA) was placed around the left main pulmonary artery. After loose apposition of the pericardial edges, the free ends of the catheters, occluder, and flow probe were threaded through the chest wall and were tunneled subcutaneously to a final position between the scapulae. A chest tube placed in the left thorax before closure was removed on the 1st postoperative day.

Morphine sulfate (10 mg, intramuscular) was administered postoperatively for pain as required. Cephazolin (1 g. intravenous) was administered intraoperatively and for 10 days postoperatively (cephalexin, 2 g/day, oral). The dogs were allowed to recover for at least 2 weeks before experimentation.

Vascular pressures were measured by attaching the fluid-filled catheters to strain-gauge manometers (P23 II), Gould Electronics, Eastlake, OH) and were referenced to atmospheric pressure with the transducers positioned at midchest at the level of the spine. Heart rate (HR) was calculated from the phasic aortic pressure trace. Left pulmonary blood flow (LQ with dot) was measured by connecting the flow probe to an electromagnetic flowmeter (SWF-4rd, Zepeda). The flow probe was calibrated in vivo on a weekly basis by the thermal dilution technique. Calibration was achieved by acutely inserting a 7 French balloon-tipped thermal dilution catheter into the pulmonary artery through a percutaneous jugular puncture after topical anesthesia (lidocaine spray). The catheter was positioned 2–3 cm beyond the pulmonic valve. The implanted perivascular hydraulic occluder was then inflated to occlude the right main pulmonary artery completely, and thus total pulmonary blood flow was directed through the left pulmonary artery (and flow probe). LQ with + was then measured by thermal dilution (9520A, American Edwards, Irvine, CA) with multiple 5-ml sterile iced injectates of 5% dextrose in water. Values for LQ with dot were referenced to body weight (milliliters per minute per kilogram).

The aortic and pulmonary artery catheters were used to obtain blood samples to measure systemic arterial and mixed venous blood gases, respectively. Systemic arterial and mixed venous pH. carbon dioxide tension, and oxygen tension were measure (ABL-3, Radiometer, Copenhagen, Denmark). Oxyhemoglobin saturation (S^O2) was measured (Hemoximeter OSM-3, Radiometer).

All solutions were prepared on the day of the experiment. Propranolol (Sigma Chemical, St. Louis, MO) was dissolved in sterile water. Phenylephrine and isoproterenol (Elkins-Sinn, Cherry Hill, NJ) were diluted in 0.9% saline. The thromboxane analogue U46619 (9,11-dideoxy-11^a9^a-epoxymethano-prostaglandin Fluorine^2a, Upjohn Laboratories, Kalamazoo, MI) was suspended in 95% ethanol and stored as a stock solution at -20 degrees Celsius. On the day of the experiment, 360 micro gram was dissolved in 60 ml of 0.9% saline.

All experiments were performed with each healthy, chronically lying on its right side in a quiet laboratory environment. Conscious dogs were not sedated. Continous LPQ with dot plots were used to assess the effects of the various pharmacologic interventions on the pulmonary circulation. LPQ with dot plots were constructed by constinuously measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure [PAP]- left atrial pressure [LAP])(PAP-LAP), and LQ with dot during gradual ([nearly equal] 1 min) inflation of the hydraulic occluder implanted around the right main pulmonary artery. This technique to measure the LPQ with dot relation is highly reproducible, and has little or no effect on systemic hemodynamics, blood gases or the zonal condition of the lung. [4].

We investigated the net effect of isoflurane anesthesia on the baseline LPQ with dot relation compared with values measured in the conscious state. For each dog (n = 12), a baseline LPQ with dot plot was first generated in the conscious state. Isoflurane anesthesia was then induced by mask and was supplemented with a subanesthetic dose of thiopental sodium (3 mg/kg, intravenous) to minimize excitatory behavior. The trachea was intubated (9 mm ID) and ventilation was controlled with a respirator (Harvard, Natick, MA) with zero end-expiratory pressure. Muscle relaxants were not used in this study. Immediately after intubation, 2% isoflurane (Anaquest, Madison, WI) was delivered by a vaporizer (Isotec 3, Ohmeda, Madison, WI). Fresh gas flow was set at 100 ml *symbol* min sup -1 *symbol* kg sup -1. Tidal volume was fixed at 15 ml/kg. Systemic arterial blood gas values were matched to values measured in the conscious state by administering supplemental oxygen (fractional inspiratory oxygen [nearly equal] 0.22) and by adjusting the respiratory rate to between 10–20 breaths/min. End-tidal carbon dioxide measured at the adapter end of the endotracheal tube was monitored continuously during the experiment (78356A, Hewlett-Packard, Andover, MA). After induction, isoflurane was allowed to equilibrate for at least 1 h to achieve steady-state conditions. This method of isoflurane anesthesia in dogs results in end-tidal isoflurance concentrations of 1.6–1.7% and 1.71.8% after 1 and 2 h, respectively, which represents approximately 1.2 MAC in dogs. [12] Plasma thiopental sodium concentration is negligible after 1 h. [13] The LPQ with dot relation was then measured as described above.

We investigated the effect of isoflurance anesthesia on the pulmonary vascular response to cumulative doses of the sympathetic alpha-adrenoreceptor agonist phenylephrine after sympathetic beta-adrenoreceptor block with propranolol. A baseline (no drug) LPQ with dot plot was first obtained in each dog (n = 6). Propranolol (1 mg/kg, intravenous) was then administered to block the beta-adrenoreceptor agonist effect of phenylephrine. This dose of propranolol completely blocks the increase in HR and the decrease in systemic arterial pressure (SAP) in response to isoproterenol. [14] LPQ with dot plots were then obtained during the cumulative administration ([nearly equal] 15 min at each dose) of phenylephrine (0.01, 0.1, 0.5, 1.0 micro gram *symbol* kg sup -1 *symbol* min intravenous). A second dose of propranolol (0.5 mg/kg, intravenous) was administered 30 min after the initial dose to ensure the efficacy of sympathetic beta-adrenoreceptor block. On a separate day, this protocol was repeated in the same dogs during isoflurane anesthesia. Anesthesia with isoflurane was induced and maintained in a manner identical to that for protocol 1.

We investigated the effect of isoflurane anesthesia on the pulmonary vascular response to cumulative doses of the sympathetic beta-adrenoreceptor agonist isoproterenol after preconstriction with the thromboxane analogue U46619. A baseline LPQ with dot plot was first obtained in each dog (n = 8). U46619 was then administered (0.14 plus/minus 0.03 micro gram *symbol* kg sup -1 *symbol min¹, intravenous) to preconstrict the pulmonary circulation before the administration of isoproterenol. LPQ with dot plots were obtained during U46619 preconstriction alone and then again with each dose of isoproterenol (0.01, 0.02, 0.05, and 0.10 micro gram *symbol* kg sup -1 *symbol* min sup -1, intravenous) during its cumulative administration ([nearly equal] 15 min at each dose) while the infusion of U46619 was continued We have verified that pulmonary vasoconstriction induced by U46619 is stable over the time course of this protocol. On a separate day, this protocol was repeated in the same dogs during isoflurane anesthesia. Anesthesia with isoflurane was induced and maintained as described in protocol 1. During isoflurane anesthesia, the dose of U46619 (0.07 plus/minus 0.01 micro gram *symbol* kg sup -1 *symbol* min sup -1, intravenous) was titrated to achieve the same level of preconstriction induced in the conscious state. This technique allowed us to assess the pulmonary vasodilator response to isoproterenol at the same level of vasomotor tone in the conscious and isoflurane-anesthetized states.

Phasic and mean vascular pressures and LQ were displayed continuously on an eight-channel strip-chart recorder (2800, Gould, Eastlake, OH). Mean pressures and LQ, measured at end-expiration, were obtained with the use of passive electronic filters with a 2-s time constant. All vascular pressures were referenced to atmospheric pressure before and after each LPQ plot. The analogue pressure and LQ signals were digitally converted and multiplexed (Medical Systems, PCM-8, Greenvale, NY) and stored on videotape (videocassette recorder AG-1260, Panasonic, Secaucus, NJ) for later playback and analysis.

The LPQ relation was linear by inspection over the empirically measured range of LQ. Therefore, linear regression analysis was used to calculate the slope and intercept for PAP-LAP (or PAP-O if LAP was less or equal to mmHg) as a function of LQ in each individual experiment. PAP-LAP intercept values were calculated at the midrange of empirically measured LQ in each protocol. This approach minimized the variance in the PAP-LAP intercept and avoided the use of intercept values outside the range of our empirical measurements; that is, PAP-LAP was not measured at LQ = 0 ml *symbol* min sup -1 *symbol* kg sup -1. The correlation coefficient for the LPQ relation in each protocol averaged 0.98 or higher. Multivariate analysis of variance in the form of Hotelling's T²was used to assess the effects of isoflurane, propranolol, phenylephrine, U46619, and isoproterenol on the regression parameters obtained in each individual experiment within each specific protocol. [15].

Student's t test for paired comparisons (protocol 1) or two-way analysis of variance (protocols 2 and 3) was used to assess the effect of isoflurane anesthesia on steady state hemodynamics and blood gases. Two-way analysis of variance was also used to assess the effects of isoflurane anesthesia on the pulmonary vascular responses to phenylephrine and isoproterenol compared with responses measured in the conscious state. The pulmonary vasodilator response to isoproterenol was expressed as the percentage decrease in U46619 preconstriction, [16] which was calculated with the following formula: Equation 1. Thus, an isoproterenol-induced decrease in PAP-LAP of 100% represents a complete reversal of U46619 preconstriction and a full return to the baseline LPQ relation. All values are presented as means plus/minus standard error.

Baseline LPQ plots for chronically instrumented dogs in the conscious state and during isoflurane anesthesia are summarized in Figure 1. Isoflurane anesthesia had no net effect on baseline PAP-LAP at any common value of LQ compared with values measured in the conscious state. Thus, isoflurane anesthesia had no net effect on the baseline LPQ relation.

Steady-state SAP was decreased and HR was increased during isoflurane anesthesia (Table 1). Systemic arterial and mixed venous blood gases during isoflurane were not significantly different from values measured in the conscious state (Table 1).

Compared with the baseline condition, propranolol had no significant effect on the baseline LPQ relation in either the conscious or isoflurane-anesthetized states (Figure 2). Phenylephrine (1.0 micro gram *symbol* kg sup -1 *symbol* min sup -1) resulted in active pulmonary vasoconstriction in both the conscious state and during isoflurane anesthesia (Figure 2). The pulmonary vascular dose-response relations for phenylephrine in the conscious and isoflurane-anesthetized states are summarized in Figure 3. Changes in PAP-LAP at LQ = 80 ml *symbol* min sup -1 *symbol* kg sup -1 from values measured during propranolol are presented. After pretreatment with propranolol, the two highest doses of phenylephrine resulted in active pulmonary vasoconstriction. Moreover, the magnitude of phenylephrine-induced pulmonary vasoconstriction was similar in the conscious state and during isoflurane anesthesia.

Propranolol had no effect on steady state hemodynamics (Table 2). Phenylephrine increased SAP, PAP, and LAP and decreased LQ and HR in both the conscious state and during isoflurane anesthesia (Table 2). Propranolol decreased mixed venous oxygen tension and S^O2in the conscious state (Table 2). Phenylephrine decreased mixed venous pH and S^O2and increased carbon dioxide tension in both the conscious and isoflurane-anesthetized states (Table 2).

Compared with the baseline condition, U46619 caused active pulmonary vasoconstriction in the conscious state and during isoflurane anesthesia (Figure 4). A smaller dose (P < 0.05) of U46619 was required during isoflurane anesthesia to match the same degree of pulmonary vascular preconstriction achieved in the conscious state. In the presence of U46619 preconstriction, isoproterenol (0.01 micro gram *symbol* kg sup -1 *symbol* min sup -1) resulted in pulmonary vasodilation in the conscious state and during isoflurane anesthesia (Figure 5). The pulmonary vascular dose-response relations for isoproterenol in the conscious and isoflurane-anesthetized states are summarized in Figure 6. After U46619 preconstriction, isoproterenol resulted in pulmonary vasodilation at all doses in both conscious and isoflurane-anesthetized dogs. However, the magnitude of the pulmonary vasolidator response to isoproterenol was markedly enhanced during isoflurane anesthesia.

U46619 increased SAP in the conscious state, and increased PAP in both the conscious state and during isoflurane anesthesia (Table 3). Isoproterenol decreased SAP and LAP in the conscious state, and increased PAP, LQ, and HR in both conditions (Table 3). U46619 decreased mixed venous S^O2in the conscious state. Isoproterenol decreased arterial and mixed venous pH during isoflurane, and decreased arterial and mixed venous carbon dioxide tension in both conditions (Table 3). Isoproterenol increased mixed venous oxygen tension and S^O2in the conscious state and during isoflurane anesthesia (Table 3).

There were three major findings in this study. First, isoflurane anesthesia had no net effect on the baseline LPQ relation. Second, the pulmonary vasoconstrictor response to the sympathetic alpha-adrenoreceptor agonist phenylephrine was not altered during isoflurane anesthesia. Third, the pulmonary vasodilator response to the sympathetic beta-adrenoreceptor agonist isoproterenol was markedly enhanced during isoflurane anesthesia. All of these studies used dogs that were chronically instrumented to measure the LPQ relation. Thus, the effects of isoflurane anesthesia on the baseline LPQ relation and the pulmonary vascular responses to sympathetic alpha- and beta-adrenoreceptor activation could be compared with responses measured in the same animals in the conscious state. Moreover, this experimental model avoids the confounding influences of acute surgical trauma, the use of background anesthetics, and the problems associated with interpreting single-point calculations of pulmonary vascular resistance.

The extent to which isoflurane anesthesia alters the baseline pulmonary circulation has been somewhat controversial. For example, previous studies that have used single-point calculations of pulmonary vascular resistance to assess the effect of isoflurane on the baseline pulmonary circulation have reported that isoflurane either increases [17,18] or has no effect [19,20] on this index of pulmonary vasomotor tone. Previous investigations that have used pulmonary vascular pressure-flow plots to assess the effect of isoflurane anesthesia on the baseline pulmonary circulation have reported that isoflurane either increases (after cyclooxygenase inhibition), [21] has no effect, [21] or decreases [22] pulmonary vasomotor tone. None of these previous studies has compared the pulmonary vascular effects of isoflurane with responses measured in the conscious state. Moreover, these acute preparations necessitated the use of background anesthetics, which could have modified the net effect of isoflurane on the baseline pulmonary circulation.

Because isoflurane is known to exert a vasodilator influence on the systemic circulation, [1] it seemed reasonable to postulate that isoflurane would also have a net vasodilator influence on the baseline pulmonary circulation. The magnitude of this effect would of necessity be very modest, because the baseline LPQ relation in conscious dogs has very little vasomotor tone. [11] Alternatively, it could be argued that isoflurane might have a net pulmonary vasoconstrictor influence on the baseline pulmonary circulation. In that regard, in vitro studies have demonstrated that isoflurane inhibits endothelium-dependent, cyclic guanosine monophosphate (cGMP)-mediated vasodilation in isolated systemic vascular smooth muscle. [23] Moreover, we have observed that endothelium-dependent cGMP-mediated pulmonary vasodilation is selectively attenuated during isoflurane anesthesia compared with the conscious state. [24] Thus, if the endogenous release of nitric oxide from endothelial cells exerts a tonic vasodilator influence on the pulmonary circulation, and if this effect is attenuated during isoflurane anesthesia, a net pulmonary vasoconstrictor response to isoflurane would be predicted. However, we have reported that nitric oxide synthase inhibition has no effect on the baseline LPQ relation in conscious dogs. [4] Thus, it was not surprising to us that isoflurane did not exert a pulmonary vasoconstrictor influence on the baseline LPQ relation in the current study.

We observed that the pulmonary vasoconstrictor response to sympathetic alpha-adrenoreceptor activation was preserved during isoflurane anesthesia compared with the response measured in the conscious state. This result is in contrast to previous that have clearly demonstrated that isoflurane attenuates the vasoconstrictor response of various systemic vascular beds to sympathetic alpha-adrenoreceptor activation. [2,3,25] These differential results may reflect regional vascular heterogeneity in terms of isoflurane's effect on alpha-adrenoreceptor activation. However, it is important to note that our results reflect the net in vivo effect of isoflurane on the pulmonary vascular response to phenylephrine. It is possible that isoflurane attenuates endogenous vasodilator mechanisms (e.g., endothelium-dependent cGMP-mediated vasodilation) that normally act to modulate the response to phenylephrine in the intact animal. This would have the effect of masking a direct attenuating effect of isoflurane on sympathetic alpha-adrenoreceptor activity.

We also observed that the pulmonary vasodilator response to sympathetic beta-adrenoreceptor activation was actually potentiated during isoflurane anesthesia compared with the response measured in the conscious state. To our knowledge, the effect of isoflurane on the pulmonary vascular response to this vasodilator stimulus has not been previously investigated. The signal transduction pathway for sympathetic beta-adrenoreceptor-mediated vasodilation involves stimulation of vascular smooth muscle adenylate cyclase, and an increase in the intracellular concentration of cyclic adenosine monophosphate (cAMP). [26] Volatile anesthetics have been shown to alter intracellular cAMP concentration. For example, isoflurane increased cAMP concentration in isolated rat aortic strips without altering cAMP phosphodiesterase activity. [3] In rat uterine homogenate, administration of halothane increased cAMP concentration, [27] and enhanced the stimulation of adenylate cyclase by isoproterenol. [28] These effects of volatile anesthetics on cAMP concentration were not altered by sympathetic beta-adrenoreceptor inhibition. [3,27,28] Moreover, halothane had no effect on beta-adrenoreceptor affinity for isoproterenol and may have even reduced beta-adrenoreceptor density. [29,30] Thus, these previous in vitro studies suggest that volatile anesthetics may enhance the action of sympathetic beta-adrenoreceptor agonists by activating adenylate cyclase in a receptor-independent manner to increase cAMP. This enhancement of cAMP-mediated vasodilation by inhalational anesthetics is in direct contrast to their inhibitory effect on cGMP-mediated vasodilation. [10,23,24,31] This inhibitory effect on cGMP-mediated pulmonary vasodilation is probably responsible for the smaller dose requirements to achieve the same degree of U46619 preconstriction during isoflurane anesthesia compared with the conscious state in protocol 3.

Baseline SAP was decreased and HR was increased during isoflurane anesthesia in all three protocols, effects that have been reported in many previous investigations. Systemic arterial and mixed venous blood gases were essentially similar in the conscious and isoflurane-anesthetized states at baseline and in response to sympathetic alpha- and beta-adrenoreceptor activation. Thus, it is unlikely that differential changes in blood gases could be responsible for any of the results in the current study.

The results of this investigation demonstrate that the effects of isoflurane anesthesia on pulmonary vasoregulation cannot be predicted by simply extrapolating from studies of the systemic circulation. Characterizing the baseline pulmonary vascular effects of isoflurane is important because of its ubiquitous clinical use. In contrast to the systemic circulation, our results suggest that sympathetic alpha-adrenoreceptor agonists may preferentially constrict the pulmonary circulation during isoflurane anesthesia. This could be deleterious in the setting of right ventricular dysfunction or pulmonary hypertension. Enhancement of sympathetic beta-adrenoreceptor-mediated pulmonary vasodilation during isoflurane anesthesia could also have significant clinical implications. This effect would be beneficial in instances where pulmonary vasodilation is desired, particularly because a smaller dose of the beta-adrenoreceptor agonist might be efficacious. It would be of interest to determine whether this effect of isoflurane anesthesia on sympathetic beta-adrenoreceptor-mediated vasodilation is also apparent in systemic vascular beds. In appears that this effect may not extend to the myocardium, because isoflurane does not increase myocardial cAMP. [32].

It is important to note that these results in dogs with a normal pulmonary vasculature may not be applicable to patients with chronic lung disease or chronic pulmonary hypertension. However, in that regard preliminary results from our laboratory indicate that isoflurane anesthesia does not alter the pulmonary vasoconstrictor response to phenylephrine in dogs with chronically increased pulmonary vascular resistance associated with left lung autotransplantation. [33].

In summary, compared with the conscious state, isoflurance anesthesia has no net effect on the baseline canine pulmonary circulation, nor does it alter the magnitude of sympathetic alpha-adrenoreceptor-mediated pulmonary vasoconstriction. In contrast, sympathetic beta-adrenoreceptor-mediated pulmonary vasodilation is potentiated during isoflurane anesthesia. These results are of potential significant clinical relevance during isoflurane anesthesia and the concomitant administration of sympathomimetic agents.

The authors thank Rosie Cousins for her outstanding technical work and Lisa DeLoriers and Ronnie Sanders for their excellent secretarial support in preparing this manuscript.