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Pulmonary Vasodilation by Ketamine Is Mediated in Part by L-Type Calcium Channels

Kaye, Alan D. MD, PhD; Banister, Ronald E. MD; Anwar, Muhammad MD; Feng, Chang J. MD; Kadowitz, Philip J. PhD; Nossaman, Bobby D. MD

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doi: 10.1213/00000539-199810000-00039
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Abstract

Ketamine is a potent analgesic at subanesthetic plasma concentrations, and its analgesic and anesthetic effects may be mediated by different mechanisms [1]. Ketamine is a potent cerebrovasodilator that produces dissociative amnesia evident on electroencephalogram as a dissociation of the thalamocortical and limbic systems [2,3]. Ketamine alters systemic arterial pressure with significant increases in heart rate, cardiac output, cardiac work, and myocardial oxygen requirements in normal humans [4,5]. The increase in blood pressure and cardiac output associated with ketamine are secondary to a direct increase in central sympathetic nervous system outflow [5]. Ketamine does not produce a significant decrease in ventilation, and the response to CO2 is maintained after an initial decrease in breathing frequency in the first 2-3 min after the induction of anesthesia. In the lung, ketamine is as effective as halothane or enflurane in preventing experimentally induced bronchospasm in dogs [6].

Studies have demonstrated various results with regard to the mechanism of action of ketamine-induced responses in the lung. The development of arginine analogs that inhibit nitric oxide synthesis have facilitated the investigation of the role of nitric oxide in regulating vascular tone and responses in the pulmonary circulation. Nitric oxide synthesis inhibitors decrease pulmonary vasodilator responses to acetylcholine, enhance pressor responses to angiotensin II and hypoxia, and have a small effect on baseline pressures in the isolated perfused rat lung, which suggests that nitric oxide plays a role in mediating or modulating pulmonary vascular responses in the rat [7-10]. In one study of rat pulmonary arteries with chronic hypoxic pulmonary hypertension, ketamine had no effect on nitric oxide-mediated relaxation in either normal or pulmonary hypertensive rats [11]. Ketamine induced responses in the rat and guinea pig via endothelium- and epithelium-independent pathways [12-15]. In the isolated rabbit portal vein, ketamine-induced vasorelaxation was not endothelium dependent but, rather, was due to blockade of the voltage-gated influx of extracellular calcium [16].

Ketamine relaxed isolated guinea pig trachea after constriction with carbachol, histamine, prostaglandin F2, and potassium [17]. One mechanism that may result in relaxation in the porcine trachealis muscle was through decreasing the excitability of postsynaptic nicotinic receptors of the intramural ganglia of the peripheral vagus nerve and the muscarinic receptors of the nerve and the smooth muscle cell [18]. In canine distal and proximal coronary arteries in vitro, relaxation responses to increasing concentrations of ketamine were observed after preconstriction with endothelin [19].

Other pathways may be involved with regard to the effects of ketamine in the lung. Ketamine-induced responses were unaffected after treatment with the cyclooxygenase inhibitor indomethacin in the trachea of guinea pigs [12]. Studies in rat and guinea pig ventricular myocytes have shown ketamine-inhibited K-ATP channel activity in single rat ventricular myocytes [20]. In isolated guinea pig ventricular myocytes treated with ketamine, there was a significant inhibition of the inward potassium rectifier current, which suggests that inhibition of this type of potassium channel may be involved in ketamine-induced responses [21]. Ketamine also decreases the frequency of open-state, long-lived, calcium-activated potassium currents in GH3 cells by a competitive interaction between calcium and ketamine [22].

Although much is known about the beneficial effects of ketamine, its mechanism in the pulmonary circulation remains controversial. Therefore, the present study was undertaken to investigate the effects of the nitric oxide synthesis inhibitor Nomega-L-nitro-L-arginine benzyl ester (L-NABE); U-37883A, an ATP-sensitive potassium channel opener; the L-type calcium channel blocker verapamil; and the cyclooxygenase blocker meclofenamate on responses to ketamine in the pulmonary vascular bed under conditions of controlled blood flow and ventilation, constant left atrial pressure in the isolated blood-perfused rat lung, and experimentally increased tone.

Methods

All experimental protocols were approved by our advisory committee for animal resources. Fifty-nine male Sprague-Dawley rats weighing 300-350 g (Hill Top Laboratories, Scottdale, PA) were anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneally. After stable anesthesia was obtained, the trachea was approached, cannulated with a short section of polyethylene tubing and connected to a rodent ventilator, and ventilated with air enriched with oxygen, a mixture of 30% O2/5% CO2, with a tidal volume of 4-5 mL/kg and 2 cm H2 O positive end-respiratory pressure. The rats were heparinized with 1000 U of heparin and rapidly exsanguinated by withdrawing blood from the carotid artery.

The lungs were exposed by median sternotomy, and a ligature was placed around the aorta to prevent systemic loss of blood. The main pulmonary artery was catheterized, and the lungs were rapidly isolated from the animal and suspended in a warmed (38[degree sign]C), humidified (100%), water-jacketed chamber. An external heat exchanger maintained the temperature of the perfusate and the isolated-lung chamber constant throughout the experiment. The perfusate solution (15 mL of heparinized blood and 5 mL of modified Krebs-Heinsleit solution) was placed into a reservoir and mixed constantly by a magnetic stirrer. The lungs were perfused with a peristaltic roller pump (Cole-Palmer Instrument Co., Berrington, IL), and blood from the left atrium was allowed to collect in the reservoir with left atrial pressure equal to atmospheric pressure. Once the isolated lung perfusion circuit was established, the flow rate was set at 8-14 mL/min to maintain physiologic baseline pulmonary arterial perfusion pressure, which averaged 15 mm Hg and was not changed during the experiment. The isolated blood-perfused lung preparation was allowed to stabilize for 30 min before beginning an experiment. All vascular pressures were measured by using transducers zeroed at the level of the pulmonary arterial cannula. Pulmonary arterial perfusion pressure, airway pressure, and reservoir blood level were continuously monitored, electronically averaged, and recorded. The modified Krebs- Heinsleit solution had the following composition (g/L): NaCl 66.37, KCl 3.58, CaCl2 2 x H2 O 3.68, KH2 PO4 1.63, MgSO4 7 x H2 O 1.45, NaHCO3 1.6, dextrose 1.0 (pH = 7.4). The solution was diluted 1:10 using double distilled water and made fresh daily.

L-NABE, acetylcholine hydrochloride, sodium arachidonate, isoproterenol hydrochloride, and sodium meclofenamate were dissolved in isotonic sodium chloride solution. Ketamine was in solution (100 mg/mL) and was diluted further in isotonic sodium chloride solution. BAY K8644 was dissolved in a 1:4 solution of cremophor EL and tris (hydroxymethyl) aminomethane (Tris) and Tris HCl, pH 7.4. The resulting suspension was warmed, and polyethylene glycol and Tris, pH 7.4, were added to make a stock solution that was stored in a brown bottle in a freezer at -20[degree sign]C. Verapamil was prepared by the manufacturer in solution. Levcromakalim was dissolved in 20% ethanol-saline solution at a concentration of 1 mg/mL and was diluted in 0.9% saline immediately before use. Nitroglycerin was prepared in a 30% alcohol/30% propylene glycol solution. The thromboxane receptor agonist U46619 (11a, 9a-epoxymethano-9a, 11b-dideoxyprostaglandin F2 alpha) was dissolved in 100% ethanol at a concentration of 10 mg/mL, and further dilutions were made in isotonic sodium chloride solution. U-37883A was sonicated in a isotonic sodium chloride solution. Working solutions were prepared frequently (every 2 or 3 days) by diluting the stock solution in isotonic sodium chloride solution, were stored in brown stoppered bottles, and were kept on ice during experiments.

Because the pulmonary vascular bed of the rat has little, if any, vasoconstrictor tone under resting conditions when the fraction of inspired oxygen is >0.21, pulmonary arterial pressure must be actively increased so that vasodilator responses can be expressed. In all the present experiments, tone was increased in the control period to an average value of 35 (31-39) mm Hg with infusion of U46619 or with ventilatory hypoxia. Under conditions of increased tone in the control period, pulmonary vascular responses of each agonist were obtained. The agonists were injected in small volumes directly into the perfusion circuit (intraarterially [IA]) distal to the pump during the control period. Afterwards, the blocker was administered and the agonists were again injected in a random sequence. Because L-NABE modestly increases tone, the U46619 infusion was initially terminated when the nitric oxide synthase inhibitor was administered and after the peak increase in pulmonary arterial pressure in response to L-NABE (100 mg/kg IA) was attained, the U46619 infusion was resumed if necessary to increase pulmonary vascular tone to a level similar to that attained during the control period. In some experiments, however, L-NABE administration alone was sufficient; in these experiments, U46619 infusion was resumed later, when pulmonary arterial pressure had decreased to <30 mm Hg.

In separate experiments with meclofenamate, verapamil, and U-37883A, responses to the agonist were again studied during the control period with U46619. Before meclofenamate, verapamil, or U-37883A administration, the U46619 infusion was terminated, and pulmonary arterial pressure was permitted to return to near control values. After the administration of verapamil (3.5 mg/kg IA) or U-37883A (2 mg/kg IA), the U46619 infusion was resumed to increase pulmonary vascular tone to a level similar to that attained during the control period. In some experiments, meclofenamate (2.5 mg/kg IA) administration alone was sufficient; in these experiments, the U46619 infusion was resumed later, when pulmonary arterial pressure had decreased <30 mm Hg.

In the last series of experiments, the effects of ketamine on the pulmonary pressor response to ventilatory hypoxia were investigated. In these experiments, the lungs were ventilated with a 3% O2/5% CO2/92% N2 gas mixture until a stable pressor response was obtained. Ketamine was then added to the perfusion reservoir to evaluate the effects of ketamine on the pressor response to hypoxia in the isolated perfused rat lung.

Arterial blood gas tensions and pH were measured at the beginning and at intervals during an experiment. Blood pH was maintained between 7.35 and 7.45 by adding small amounts of NaHCO3 solution to the perfusion reservoir. To serve as a control before agonist injections, equivalent volumes of vehicle were injected directly into the perfusion circuit. All injections were made in small volumes in a random sequence, and sufficient time was permitted between agonist injections for pressures to return to baseline values. Because pulmonary blood flow and outflow pressure were maintained constant, changes in perfusion pressure in this preparation reflect changes in pulmonary vascular resistance. All vascular pressures are expressed in absolute units (mm Hg) as mean +/- SEM. The data were analyzed using analysis of variance with post hoc Scheffe's F-test for repeated measures (StatView Co., Abascus Concepts, Berkeley, CA) on a computer. A P value of <0.05 was used as the criterion for statistical significance.

Results

Because changes in pulmonary blood flow induce passive changes in pulmonary vascular resistance, the direct effects of ketamine on the pulmonary vascular bed were investigated in the rat under constant-flow conditions. Under low resting tone conditions, injections of ketamine in doses of 0.01-1.0 mg produced only small decreases in pulmonary arterial pressure (Figure 1). However, when baseline tone in the pulmonary vascular bed was increased to a high steady value with an infusion of U46619 or in response to ventilatory hypoxia, injecting 0.01-1.0 mg of ketamine into the perfusion circuit caused significant dose-related decreases in pulmonary arterial perfusion pressure (Figure 1). Decreases in pulmonary arterial pressure in response to ketamine were rapid in onset, and pressure returned to control values over 3-5 min, depending on the dose injected.

Figure 1
Figure 1:
Influence of ketamine under low tone (upper) and conditions of increased tone with U46619 (middle) or ventilatory hypoxia (lower). *Changes are significant (P < 0.05).

When baseline tone in the pulmonary vascular bed was increased to a high steady value with an infusion of U46619, injections of ketamine (30-300 [micro sign]g IA), acetylcholine (100-1000 ng IA), nitroglycerin (0.3-3 ng IA), and isoproterenol (0.03-0.3 ng IA) into the perfusion circuit caused significant dose-related decreases in pulmonary arterial pressure (Figure 2). Decreases in pulmonary arterial pressure in response to IA injections of acetylcholine were significantly attenuated after the administration of L-NABE (Figure 2). Decreases in pulmonary arterial pressure in response to IA injections of ketamine, nitroglycerin, and isoproterenol were not significantly changed after the administration of L-NABE (Figure 2).

Figure 2
Figure 2:
Influence of Nomega-L-nitro-L-arginine benzyl ester (L-NABE) on vasodilator responses to ketamine, isoproterenol, acetylcholine, and nitroglycerin under conditions of increased tone with U46619 infusion. *Changes are significant (P < 0.05).

When baseline tone in the pulmonary vascular bed was increased to a high steady value with an infusion of U46619, injecting 10-100 [micro sign]g of ketamine and levcromakalim into the perfused pulmonary artery caused significant dose-related decreases in pulmonary arterial pressure (Figure 3). Decreases in pulmonary arterial pressure in response to IA injections of ketamine were not significantly attenuated after the administration of U-37883A, whereas the vasodilator responses to the K (+-ATP) channel opener levcromakalim were significantly reduced (Figure 3).

Figure 3
Figure 3:
Influence of U-37883A on vasodilator responses to ketamine and levcromakalim under increased tone conditions. *Changes are significant (P < 0.05).

Meclofenamate significantly attenuated vasodepressor responses to arachidonic acid (Figure 4). Decreases in pulmonary arterial pressure in response to injections of ketamine were not significantly attenuated after the administration of the cyclooxygenase inhibitor meclofenamate (Figure 4).

Figure 4
Figure 4:
Influence of meclofenamate on vasodilator responses to ketamine and arachidonic acid. *Changes are significant (P < 0.05).

Verapamil significantly decreased BAY K8644-induced pressor responses in the pulmonary vascular bed of the rat (Figure 5). When pulmonary arterial pressure was increased by the infusion of U46619, decreases in pulmonary arterial pressure in response to IA injections of ketamine were significantly smaller than responses obtained when ketamine was injected during the control period when tone was increased with an U46619 infusion (Figure 5). Decreases in pulmonary arterial pressure in response to injections of nitroglycerin and levcromakalim were not significantly attenuated after the administration of the calcium channel blocker verapamil (Figure 5).

Figure 5
Figure 5:
Influence of verapamil on responses to ketamine, nitroglycerin, levcromakalim, and BAY K 8644. *Changes are significant (P < 0.05).

Discussion

Our results show that ketamine decreases pulmonary arterial pressure and that responses were attenuated after the administration of the L-type calcium channel antagonist when tone in the pulmonary vascular bed was increased to a high steady level with U46619. Pulmonary blood flow and left atrial pressure were maintained constant so that decreases in pulmonary pressure reflected decreases in pulmonary arterial vascular resistance. The decreases in pulmonary arterial pressure were dose-dependent and were not reduced by L-NABE, meclofenamate, or U-37883A. Decreases in pulmonary vascular resistance in response to ketamine seem to be independent of the release of endothelium-derived nitric oxide, the release of vasodilator products in the cyclooxygenase pathway, or the activation of ATP-sensitive potassium channels.

Although the exact mechanism by which ketamine induces a vasodilator effect in the pulmonary vascular bed is not well understood, the results of the present study suggest that this induction drug has significant pulmonary vasodilator activity at clinically relevant doses. Ketamine produced dose-related decreases in perfusion pressure in the rat pulmonary vascular bed that were not significantly decreased in the presence of meclofenamate, which suggests a minimal role for vasodilator prostaglandins. The observation that vasodilator responses to ketamine are not inhibited by L-NABE and meclofenamate suggests that the release of nitric oxide and the formation of cyclooxygenase products are probably not major mechanisms of action.

The effects of ketamine may involve an effect on calcium influx or release from the sarcoplasmic reticulum. In rabbit and guinea-pig preparations, ketamine inhibits L-type calcium channels, reduces calcium influx, and/or activates a calcium-ATPase [23-25]. However, in vascular smooth muscle cells, ketamine had no significant effect on calcium uptake into intracellular stores or on calcium extrusion [26]. Ketamine was also found to inhibit agonist-induced synthesis of inositol 1,4,5-triphosphate in rabbit mesenteric artery and inhibit the release of calcium [27]. A similar mechanism for the inhibition of calcium release was found in the rabbit femoral artery, in which ketamine decreased the release of calcium and reduced phospholipase C activity [28].

In studies in GH3 cells, calcium-activated potassium channels were blocked and calcium levels decreased in a dose-dependent manner [29]. A follow-up study in these cells indicated a significant disruption of the phospholipase A2 arachidonic acid signal transduction pathway [30]. In the present study, vasodilator responses to ketamine were not altered after the administration of the ATP-sensitive potassium channel blocker U-37883A in a dose that significantly decreased responses to the ATP-sensitive potassium channel opener levcromakalim, which suggests that ketamine-induced vasodilation does not involve activation of these potassium channels.

The results of the present investigation suggest that calcium is involved in ketamine responses in the lung because ketamine-induced vasodilator responses were attenuated by the L-type calcium channel blocker verapamil. Verapamil inhibited responses to BAY K 8644, a nifedipine analog that promotes calcium entry, which shows that L-type calcium channels were blocked. Additional mechanisms for the ketamine-induced vasodilator response may play a role in mediating responses in the pulmonary vascular bed of the rat.

The use of L-arginine analogs, such as L-NABE, is complicated because some studies have demonstrated potentiation of vasoconstrictor response to U-46619 and have questioned the use of these probes in studies on the role of endothelium-derived relaxing factor in the pulmonary vascular bed in vivo [7,8]. We have, however, used Nomega-L-nitro-L-arginine methyl ester, L-NABE, and Nomega-L-nitro-L-arginine with very similar results [9,10]. Further, the data in the present investigation show that vasodilator responses to ketamine were similar when U-46619 or hypoxia were used to increase tone, which suggests that an interaction of ketamine with U46619 is minimal under the conditions of the present experiments.

In conclusion, the results of the present study show that ketamine has significant vasodilator activity and that responses were attenuated after the administration of the L-type calcium channel blocker verapamil when tone in the pulmonary vascular bed was increased to a high steady level. The vasodilator response to ketamine seems to be independent of the release of endothelial-derived nitric oxide or cyclooxygenase products in the rat pulmonary vascular bed. These data also demonstrate that vasodilator responses to ketamine do not involve the activation of ATP-sensitive potassium channels.

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