Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca²⁺Sensitivity in Pulmonary Veins

All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation (Cleveland, Ohio).

Healthy male mongrel dogs weighing approximately 28 kg were anesthetized with intravenous pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 μg/kg). After tracheal intubation, the lungs were mechanically ventilated. A catheter was placed in the right femoral artery, and the dogs were exsanguinated by controlled hemorrhage. A left lateral thoracotomy was performed through the fifth intercostal space, and the heart was arrested with induced ventricular fibrillation. The heart and lungs were removed from the thorax en bloc , and the lower right and left lung lobes were dissected free. Intralobar PVs (third generation, 1–2 mm ID) were carefully dissected and immersed in cold modified Krebs-Ringer’s bicarbonate (KRB) solution composed of 118.3 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO⁴, 1.2 mm KH²PO⁴, 2.5 mm NaHCO³, 0.016 mm Ca-EDTA, and 11.1 mm glucose. PVs were cleaned of connective tissue and cut into ring segments 4–5 mm in length, with special care taken not to damage the endothelium. In some rings, the endothelium was intentionally removed by gently rubbing the intimal surface with a cotton swab. The integrity of the endothelium was verified by assessing the vasorelaxant response to the endothelium-dependent vasodilator, bradykinin (10⁻⁸m)4,17,18during acetylcholine contraction. Bradykinin induced more than 20% relaxation in endothelium-intact (E+) PV rings and no relaxation or a slight contraction in endothelium-denuded (E−) PV rings.

Pulmonary vein rings were vertically mounted between two stainless steel hooks in organ baths filled with 25 ml KRB solution (37°C) gassed with 95% oxygen and 5% carbon dioxide. One of the hooks was anchored, and the other was connected to a strain gauge to measure isometric force. The rings were stretched at 5-min intervals in increments of 0.5 g to achieve optimal resting tension. Optimal resting tension was defined as the minimum amount of stretch required to achieve the largest contractile response to 60 mm KCl and was determined in preliminary experiments to be 1.5 g. After the PV rings had been stretched to their optimal resting tension, the contractile response to 60 mm KCl was assessed. After washout of KCl from the organ chamber and the return of isometric tension to prestimulation values, a concentration–response curve to acetylcholine was performed in each ring under baseline tone conditions (i.e. , no precontraction). This was achieved by increasing the concentration of acetylcholine in half-log increments (from 10⁻⁸m to 10⁻⁵m) after the response to each preceding concentration had reached a steady state.

The effects of ketamine (10⁻⁵to 10⁻³m) on the acetylcholine concentration-response relation were assessed in E+ and E− PV rings. Ketamine was directly added to the organ bath 30 min before acetylcholine contraction. The contractile responses to acetylcholine in ketamine-pretreated rings were compared with responses in matched untreated rings.

To determine whether the nitric oxide pathway is involved in the ketamine-induced attenuation of acetylcholine contraction, E+ PV rings were pretreated for 30 min with N -nitro-l-arginine methylester (l-NAME 10⁻⁴m), an inhibitor of nitric oxide synthase, alone or in combination with ketamine (10⁻⁴m). The contractile responses to acetylcholine in l-NAME–pretreated rings were compared with responses in untreated rings and rings pretreated with l-NAME combined with ketamine.

To determine whether Ca²⁺influx though L-type VOCCs and/or IP³-mediated Ca²⁺release was involved in the ketamine-induced attenuation of acetylcholine contraction, E− PV rings were pretreated for 30 min with nifedipine (10⁻⁵m), an inhibitor of L-type VOCCs, or 2-aminoethoxydiphenylborate (2-APB; 10⁻⁴m), an inhibitor of IP³-mediated Ca²⁺release, alone or in combination with ketamine (10⁻⁴m). The contractile responses to acetylcholine after pretreatment with the inhibitors were compared to responses in untreated rings and rings pretreated with the inhibitors combined with ketamine.

Intralobar PVs (2–4 mm ID) were dissected carefully and immersed in cold modified KRB solution. The PVs were cleaned of connective tissue and cut into strips (2 × 6 mm). The endothelium was removed by gently rubbing the intimal surface with a cotton swab. Endothelial denudation was later verified by the absence of a vasorelaxant response to bradykinin (10⁻⁸m).

Intralobar PV strips without endothelium were loaded with 5 × 10⁻⁶m acetoxylmethyl ester of fura-2 (fura-2/AM) solution. A noncytotoxic detergent, 0.05% cremophor EL, was added to solubilize the fura-2/AM in the solution. After fura-2 loading, the PV strips were washed with KRB buffer to remove uncleaved fura-2/AM and were mounted between two stainless steel hooks in a temperature-controlled (37°C) 3-ml cuvette. The strips were continuously perfused at 12 ml/min with the KRB solution bubbled with 95% oxygen and 5% carbon dioxide (pH 7.4). One hook was anchored, and the other was connected to a strain gauge transducer (Grass FTO³; Grass Instrument Co., Quincy, MA) to measure isometric tension. The resting tension was adjusted to 0.5 g, which was determined in preliminary studies to be optimal for achieving a maximum contractile response to 40 mm KCl. We used 40 mm KCl rather than 60 mm KCl in the strip studies because the higher concentration was associated with a prolonged washout period before tension and intracellular Ca²⁺concentration ([Ca²⁺]ⁱ) returned to baseline values. Fluorescence measurements were performed using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Because calculations of absolute concentration of [Ca²⁺]ⁱrely on a number of assumptions, the 340-to-380 fluorescence ratio (340/380 ratio) was used as a measure of [Ca²⁺]ⁱ. The individual 340 and 380 signals were also measured in all experiments, and the signals were observed to change in opposite directions in response to the various interventions. Fluorescence unrelated to fura-2 consists of two parts: background fluorescence and tissue autofluorescence. Background fluorescence is essentially constant. Moreover, each PV strip served as its own control. Therefore, background fluorescence was assumed to be constant and was not subtracted from the calculated 340/380 ratio. However, tissue autofluorescence can be large and variable.19,20One way to minimize this effect is to load the PV strip with fura-2 to a level well above the initial autofluorescence level. In the current study, the fluorescence intensity after loading fura-2 was approximately three times that of the initial autofluorescence. Another way to solve this potential problem is to choose proper experimental conditions. Before the PV strips were loaded with fura-2, we measured the autofluorescence (emission at 510 nm) by taking individual analog measurements at 340 and 380 nm at a temperature of 37°C. The autofluorescence of PV strips was constant under these conditions. The temperature of all solutions was maintained at 37°C in a water bath. Fura-2 fluorescence signals (340 and 380 nm and 340/380 ratio) and tension were measured at a sampling frequency of 2 Hz and were collected with a software package from Photon Technology International.

We measured tension and [Ca²⁺]ⁱsimultaneously to investigate whether ketamine alters the [Ca²⁺]ⁱ–tension relation in E− PV strips. In protocol 1, PV strips were treated with 40 mm KCl. After changes in [Ca²⁺]ⁱand tension had reached new steady state values (10 min), the strips were washed with fresh KRB solution, and both tension and [Ca²⁺]ⁱreturned to baseline. After a return to baseline, the strips were treated with a Ca²⁺-free buffer containing 2 mm EGTA for 10 min. This solution was replaced with a Ca²⁺-free buffer that did not contain EGTA. After 10 min, this solution was replaced with a Ca²⁺-free solution containing 40 mm KCl. Finally, after 10 min, the extracellular Ca²⁺concentration was increased in control and ketamine (10⁻⁴m)–pretreated strips in an incremental fashion from 0 to 0.125, 0.25, 0.5, 1.25, and 2.5 mm. In protocol 2, the same procedure was repeated in strips pretreated with acetylcholine (10⁻⁶m), alone or in combination with ketamine. In protocol 3, the same procedure was repeated in strips pretreated with acetylcholine (10⁻⁶m) and inhibitors of either protein kinase C (PKC) (bisindolylmaleimide I [BIS1; 3 × 10⁻⁶m]) or rho-kinase (ROK) (Y27632; 10⁻⁶m), alone or in combination with ketamine (10⁻⁴m). PV strips were pretreated with ketamine, acetylcholine, the various inhibitors, or their combination for 15 min.

All drugs were of the highest purity commercially available: acetylcholine, l-NAME, nifedipine, 2-APB, BIS1, cremophor EL, dimethyl sulfoxide (Sigma, St. Louis, MO), Y-27632 (Calbiochem-Novabiochem Corp, San Diego, CA), and fura-2/AM (Texas Fluorescence Labs, Austin, TX). BIS1 and fura-2/AM were dissolved in dimethyl sulfoxide and diluted with distilled water. The final concentration of dimethyl sulfoxide in the organ bath and cuvette was less than 0.1% (volume/volume). None of the agents or solutions caused significant shifts in isometric tension or the 340/380 ratio at the concentrations used in these studies.

All data are expressed as mean ± SD. Contractile responses to acetylcholine are expressed as the percentage contraction induced by 60 mm KCl in the ring studies and 40 mm KCl in the strip studies. The acetylcholine contractile responses were compared in matched control and “treated” (ketamine and various inhibitors) rings or strips from the same dog. The [Ca²⁺]ⁱ–tension relation was calculated and plotted using Sigmaplot 8.0 (SPSS Inc., Chicago, IL). Statistical analysis was performed using two-way repeated-measures analysis of variance with SPSS for Windows software (version 11.5; SPSS Inc.). When differences among groups were detected, post hoc  analysis utilized the least significant difference test. For the ring studies, acetylcholine dose was used as the within-subject factor, and treatment (with or without) was used as the between-subject factor. For the strip studies, Ca²⁺concentration was used as the within-subject factor, and treatment (with or without) was used as the between-subject factor. A P  value of less than 0.05 was chosen as statistically significant. In all experiments, sample size (n values) equal the number of dogs from which PV rings or strips were taken.

Ketamine had no effect on resting tension. Ketamine attenuated acetylcholine contraction in a dose-dependent manner (P < 0.001; fig. 1). Ketamine (10⁻⁵m) had no effect (E+: P = 0.774; E−P = 0.641) on the acetylcholine dose–response relation, whereas ketamine (10⁻⁴m and 10⁻³m) attenuated acetylcholine contraction in both E+ rings (P = 0.001 and P < 0.001, respectively; fig. 1A) and E− rings (P = 0.001 and P < 0.001, respectively; fig. 1B).

We tested the hypothesis that the ketamine-induced attenuation of acetylcholine contraction involves the nitric oxide pathway. l-NAME had no effect on resting tension. Pretreatment of E+ rings with l-NAME markedly potentiated (P < 0.001) acetylcholine contraction (fig. 2). The ketamine-induced attenuation of acetylcholine contraction was still observed after pretreatment with l-NAME (P = 0.002; fig. 2). These results indicate that endothelium-derived nitric oxide acts to modulate acetylcholine contraction, but the ketamine-induced attenuation of acetylcholine contraction does not require the nitric oxide signaling pathway.

We tested the hypothesis that the ketamine-induced attenuation of acetylcholine contraction is mediated by effects on either Ca²⁺influx through L-type VOCCs and/or IP³-mediated Ca²⁺release. Neither nifedipine nor 2-APB had an effect on resting tension. Pretreatment of E− rings with nifedipine (fig. 3A) or 2-APB (fig. 3B) attenuated acetylcholine contraction (P < 0.001 and P < 0.001, respectively). However, the ketamine-induced attenuation of acetylcholine contraction was still observed after pretreatment with nifedipine (P < 0.001; fig. 3A) or 2-APB (P = 0.021; fig. 3B). These results indicate that both Ca²⁺influx via  L-type VOCCs and IP³-mediated Ca²⁺release mediate a component of acetylcholine contraction, but neither of these pathways are required for the ketamine-induced attenuation of acetylcholine contraction in PVs.

To assess the effects of ketamine on PV myofilament Ca²⁺sensitivity, control and ketamine (10⁻⁴m)–pretreated E− PV strips bathed in a Ca²⁺-free buffer containing 40 mm KCl were exposed to incremental increases in extracellular Ca²⁺concentration. Increasing extracellular Ca²⁺concentration resulted in virtually identical increases in [Ca²⁺]ⁱ(P = 0.869; fig. 4A) and tension (P = 0.875; fig. 4B) in control and ketamine-pretreated strips. Thus, ketamine had no effect (P = 0.892) on the [Ca²⁺]ⁱ–tension relation (fig. 4C), which suggests that ketamine alone had no effect on myofilament Ca²⁺sensitivity.

Because ketamine alone had no effect on PV myofilament Ca²⁺sensitivity, we tested the hypothesis that ketamine decreases myofilament Ca²⁺sensitivity in the presence of acetylcholine. Increasing extracellular Ca²⁺resulted in greater increases (P = 0.004) in tension in acetylcholine-pretreated (10⁻⁶m) strips compared with control (fig. 5A), whereas increases in [Ca²⁺]ⁱwere similar (P = 0.836) in control and acetylcholine-pretreated strips (fig. 5B). As a result, acetylcholine caused a leftward shift (P = 0.003) in the [Ca²⁺]ⁱ–tension relation (fig. 5C), such that for a given value of [Ca²⁺]ⁱ, tension was greater in acetylcholine-pretreated E− PV strips compared with control (i.e. , acetylcholine increased myofilament Ca²⁺sensitivity). Ketamine (10⁻⁴m) attenuated the acetylcholine-induced increase in tension (P = 0.034) but had no effect on [Ca²⁺]ⁱ(P = 0.836). This resulted in a rightward shift in the [Ca²⁺]ⁱ–tension relation in acetylcholine-pretreated strips (P = 0.038; fig. 5C), which suggests that ketamine attenuates the acetylcholine-induced increase in PV myofilament Ca²⁺sensitivity.

We tested the hypothesis that the ketamine-induced attenuation of the increase in PV myofilament Ca²⁺sensitivity in response to acetylcholine involves the ROK signaling pathway. The effect of ROK inhibition, alone or in combination with ketamine, on the [Ca²⁺]ⁱ–tension relation was assessed in acetylcholine-pretreated E− PV strips. ROK inhibition attenuated the acetylcholine-induced increase in tension (P < 0.001; fig. 6A) but had no effect on [Ca²⁺]ⁱ(P = 0.843; fig. 6B). As a result, ROK inhibition caused a rightward shift (P = 0.001) in the [Ca²⁺]ⁱ–tension relation in acetylcholine-pretreated PVs (fig. 6C). In the presence of ROK inhibition in acetylcholine-pretreated PVs, ketamine decreased tension (P = 0.034; fig. 6A) but not [Ca²⁺]ⁱ(P = 0.843; fig. 6B). As a result, ketamine still caused a rightward shift in the [Ca²⁺]ⁱ–tension relation (P = 0.039; fig. 6C). These results indicate that the acetylcholine-induced increase in myofilament Ca²⁺sensitivity involves the ROK signaling pathway. However, the ROK signaling pathway is not required for the ketamine-induced attenuation of the increase in myofilament Ca²⁺sensitivity in response to acetylcholine in PVs.

We tested the hypothesis that the ketamine-induced attenuation of the increase in myofilament Ca²⁺sensitivity in response to acetylcholine is due to an effect on the PKC signaling pathway. PKC inhibition attenuated the acetylcholine-induced increase in tension (P < 0.001; fig. 7A) but had no effect on [Ca²⁺]ⁱ(P = 0.193; fig. 7B). As a result, PKC inhibition caused a rightward shift (P < 0.001) in the [Ca²⁺]ⁱ–tension relation in acetylcholine-pretreated PVs (fig. 7C). In the presence of PKC inhibition in acetylcholine-pretreated PVs, ketamine had no effect on either tension (P = 0.607; fig. 7A) or [Ca²⁺]ⁱ(P = 0.193; fig. 7B). Thus, the ketamine-induced decrease in myofilament Ca²⁺sensitivity in acetylcholine-pretreated PVs was abolished (P = 0.798; fig. 7C), which suggests that the PKC signaling pathway mediated this effect of ketamine.

Interest in PV has increased recently after the report that a number of patients with atrial fibrillation have an ectopic electrical focus originating within the PVs.21PVs are a primary site for entry of vagal nerves into the left atrium.22The parasympathetic neurotransmitter, acetylcholine, is a muscarinic receptor agonist that has been reported to cause both PV relaxation23as well as PV contraction,3,4,13,14depending on the concentration of acetylcholine, the level of vasomotor tone, the muscarinic receptor subtype, and the species studied. In the current study, we observed that acetylcholine caused dose-dependent contraction in E+ PVs under baseline tone conditions (i.e. , no precontraction). Acetylcholine contraction was potentiated by removing the endothelium and by inhibition of nitric oxide synthase. Acetylcholine contraction was attenuated by inhibiting L-type Ca²⁺channel entry and IP³-mediated Ca²⁺release from the sarcoplasmic reticulum. Acetylcholine increased myofilament Ca²⁺sensitivity, and this effect involved both the PKC and ROK signaling pathways. Taken together, these results indicate that acetylcholine-induced PV contraction is mediated by both an increase in [Ca²⁺]ⁱand myofilament Ca²⁺sensitivity. Conversely, acetylcholine contraction is modulated by endothelium-derived nitric oxide.

The effect of ketamine on acetylcholine-induced changes in PV tone has not been previously investigated. Ketamine has been reported to cause pulmonary vasodilation in precontracted isolated lungs of the cat24and rat25and to induce vasorelaxation in precontracted pulmonary arterial rings of the rabbit,26rat,27and guinea pig.28In all of these studies, the pulmonary vascular effects of ketamine were found to be endothelium independent. In contrast, our laboratory has reported that ketamine attenuates the pulmonary artery relaxation response to acetylcholine by inhibiting both the nitric oxide– and endothelium-derived hyperpolarizing factor components of the response.16In the current study, we tested the hypothesis that the ketamine-induced attenuation of acetylcholine contraction was mediated by an inhibitory effect of ketamine on an endothelium-derived contracting factor (e.g. , thromboxane). However, we observed that removing the endothelium actually potentiated acetylcholine contraction. This effect was likely due to the loss of the modulating influence of nitric oxide on acetylcholine contraction, because inhibition of nitric oxide synthase also potentiated acetylcholine contraction. Moreover, ketamine attenuated acetylcholine contraction in E− PVs as well as in PVs pretreated with the nitric oxide synthase inhibitor l-NAME. Therefore, the ketamine-induced inhibition of acetylcholine contraction does not involve an effect on endothelium-derived contracting factors or nitric oxide.

Vascular smooth muscle contraction is initiated by an increase in [Ca²⁺]ⁱ. This results from an influx of Ca²⁺across the sarcolemma through plasma membrane channels (e.g. , VOCCs) as well as Ca²⁺release from the sarcoplasmic reticulum (e.g. , IP³-mediated Ca²⁺release). Electrophysiologic and pharmacologic studies suggest that there are at least six types of VOCCs (types L, T, N, R, Q, and P).29At least two types of VOCCs are present in vascular smooth muscle: “transient” (T-type) channels and “long-lasting” (L-type) channels.30,31In most vascular smooth muscle cells, L channels are more numerous and probably are the most important route of calcium influx.32,33T channels are relatively resistant to inhibition by dihydropyridine antagonists (nifedipine and its derivatives), whereas L channels are highly sensitive to dihydropyridine antagonists.34,35Because L channels are the most important route of calcium influx, we only investigated the role of L-type VOCCs in the ketamine-induced attenuation of acetylcholine contraction. Acetylcholine contraction was inhibited by nifedipine and 2-APB, which indicates that acetylcholine contraction of PVs involves both Ca²⁺influx through L-type VOCCs and IP³-mediated Ca²⁺release. Ketamine has previously been shown to inhibit transmembrane Ca²⁺influx in rabbit and guinea pig cardiac muscle36,37as well as canine airway smooth muscle.38In addition, ketamine has been reported to decrease IP³formation in response to norepinephrine in neonatal rat cardiomyocytes.39In the current study, ketamine still attenuated acetylcholine contraction in PV pretreated with nifedipine or 2-APB, which indicates that neither L-type VOCCs nor IP³-mediated Ca²⁺release is involved in this response.

In addition to changes in [Ca²⁺]ⁱ, vascular smooth muscle contractility depends on the Ca²⁺sensitivity of the contractile apparatus (i.e. , myofilament Ca²⁺sensitivity). Muscarinic receptor activation results in increased levels of 1,2-diacylglycerol via  hydrolysis of membrane-associated phospholipase C, which in turn activates the Ca²⁺- and lipid-dependent enzyme PKC.40When activated, PKC may directly or indirectly inhibit myosin light chain phosphatase,40,41thereby increasing regulatory myosin light chain phosphorylation and force for a given [Ca²⁺]ⁱ.42Agonist-induced activation of the ROK signaling pathway can also increase myofilament Ca²⁺sensitivity by inhibiting myosin light chain phosphatase.43However, only two studies have investigated agonist-induced changes in myofilament Ca²⁺sensitivity in PVs.5,7PKC was shown to mediate sustained contraction in response to endothelin,7whereas norepinephrine and thromboxane A²increased Ca²⁺sensitivity via  the ROK and tyrosine kinase signaling pathways.5In the current study, acetylcholine increased PV myofilament Ca²⁺sensitivity, as reflected by the acetylcholine-induced leftward shift in the [Ca²⁺]ⁱ–tension relation. Both PKC inhibition and ROK inhibition attenuated the acetylcholine-induced increase in myofilament Ca²⁺sensitivity, suggesting that these signaling pathways are involved in the response. PKC activation has been reported to be involved in acetylcholine-induced increases in myofilament Ca²⁺sensitivity in rabbit aorta, rabbit bladder, and human bladder.44Ketamine alone had no effect on the [Ca²⁺]ⁱ–tension relation, whereas it caused a rightward shift in the relation in PVs pretreated with acetylcholine. This effect of ketamine was still observed after ROK inhibition but was abolished after PKC inhibition. These results suggest that the ketamine-induced inhibition of acetylcholine contraction is due to a decrease in myofilament Ca²⁺sensitivity and that this effect is mediated by the PKC pathway.

We are aware of only two studies that have investigated the effect of ketamine on myofilament Ca²⁺sensitivity. Akata et al.  45reported that ketamine had no effect on myofilament Ca²⁺sensitivity in either membrane-permeabilized or membrane-intact rat mesenteric resistance arterial strips. Hanazaki et al.  46reported that ketamine (2 × 10⁻⁴m) had no effect on myofilament Ca²⁺sensitivity in the presence or absence of muscarinic receptor stimulation (acetylcholine [10⁻⁵m]) in canine tracheal smooth muscle. Moreover, that same group demonstrated that PKC has little or no role in regulating Ca²⁺sensitivity during muscarinic stimulation (acetylcholine) in canine tracheal smooth muscle,47which likely explains why ketamine had no effect on Ca²⁺sensitivity in the presence of acetylcholine in their previous study.46We also observed that ketamine alone had no effect on the [Ca²⁺]ⁱ–tension relation in PVs, although ketamine attenuated the acetylcholine-induced increase in Ca²⁺sensitivity via  an effect on the PKC pathway. Therefore, canine tracheal smooth muscle and PVs are distinct in terms of the role of PKC in the response to acetylcholine. Ketamine only exerts an effect on the acetylcholine response when the PKC pathway is activated.

The effect of ketamine on pulmonary vascular resistance is controversial. Ketamine increased pulmonary vascular resistance in patients with preexisting pulmonary hypertension,48whereas the opposite effect was reported in critically ill and acutely traumatized patients.49We acknowledge that results obtained from our study must be carefully extrapolated to clinical practice. However, because pulmonary venous resistance is an important component of total pulmonary vascular resistance,1our results provide new insight concerning the effect of ketamine on pulmonary venous tone.

The plasma concentration of ketamine after intravenous administration of 2 mg/kg has been reported to be 1.1 × 10⁻⁴m.50Ketamine at a concentration of 10⁻⁴m attenuated the acetylcholine contractile response in PVs, so this effect is apparent at a clinically relevant concentration.

In summary, ketamine attenuates acetylcholine contraction in PVs by inhibiting the acetylcholine-induced increase in myofilament Ca²⁺sensitivity. This inhibitory effect of ketamine on acetylcholine contraction in PVs involves the PKC signaling pathway.