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Meeting Abstracts  |   March 2005
Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca2+Sensitivity in Pulmonary Veins
Author Affiliations & Notes
  • Xueqin Ding, M.D., Ph.D.
    *
  • Derek S. Damron, Ph.D.
  • Paul A. Murray, Ph.D.
  • * Research Fellow, † Assistant Professor, ‡ Professor.
Article Information
Meeting Abstracts   |   March 2005
Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca2+Sensitivity in Pulmonary Veins
Anesthesiology 3 2005, Vol.102, 588-596. doi:
Anesthesiology 3 2005, Vol.102, 588-596. doi:
PULMONARY venous resistance is an important component of total pulmonary vascular resistance.1 Although a great deal is known about the regulation of pulmonary arterial vasomotor tone, comparatively little is known about pulmonary venous tone. Pulmonary veins (PVs) are known to constrict and dilate in response to a number of stimuli.2–4 However, there are only a handful of studies that have investigated cellular mechanisms that regulate pulmonary venous tone.5–8 Moreover, there is only one study in the literature that has assessed the effects of anesthetics on pulmonary venous tone,9 and that study did not address cellular mechanisms of action of anesthetics.
Acetylcholine is a muscarinic receptor agonist that has an important role in the regulation of pulmonary vasomotor tone.10,11 Acetylcholine causes endothelium-dependent vasodilation in pulmonary arteries12 but can induce species-dependent vasoconstriction in PVs.3,4,13,14 In previous studies in canine PVs,15 we observed that acetylcholine causes contraction that is modulated by nitric oxide and partially mediated by metabolites of the cyclooxygenase pathway and involves Ca2+influx through voltage-operated Ca2+channels (VOCCs), inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release from the sarcoplasmic reticulum, and an increase in myofilament Ca2+sensitivity.
Ketamine is an intravenous anesthetic that is often used as an induction agent in patients with hemodynamic instability. We previously reported that ketamine attenuated the vasorelaxant response to acetylcholine in isolated canine pulmonary artery.16 However, the extent to which ketamine alters the PV response to acetylcholine is entirely unknown. We tested the hypothesis that ketamine exerts an inhibitory effect on acetylcholine-induced contraction in PVs. Specifically, we assessed the roles of the endothelium, Ca2+influx and release, and myofilament Ca2+sensitivity on ketamine-induced changes in acetylcholine contraction.
Materials and Methods
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation (Cleveland, Ohio).
Preparation of Pulmonary Venous Rings
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)4,17,18 during 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.
Isometric Tension Experiments
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.
Preparation of PVSM Strips
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).
Simultaneous Measurement of Tension and [Ca2+]i
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.19,20 One 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.
PV Strip Experimental Protocols
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.
Solutions and Chemicals
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.
Statistical Analysis
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.
Results
Effect of Ketamine on Acetylcholine Contraction
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).
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
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Role of Nitric Oxide in Ketamine-induced Attenuation of Acetylcholine Contraction
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.
Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
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Role of L-type Ca2+Channel Influx and IP3-mediated Ca2+Release in Ketamine-induced Attenuation of Acetylcholine Contraction
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.
Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
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Effect of Ketamine on PV Myofilament Ca2+Sensitivity
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.
Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
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Effect of Ketamine on PV Myofilament Ca2+Sensitivity in the Presence of Acetylcholine
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.
Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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Role of the Rho-Kinase Signaling Pathway in Ketamine-induced Attenuation of 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.
Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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Role of the Protein Kinase C Signaling Pathway in Ketamine-induced Attenuation of Myofilament Ca2+Sensitivity
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.
Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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Discussion
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.21 PVs are a primary site for entry of vagal nerves into the left atrium.22 The parasympathetic neurotransmitter, acetylcholine, is a muscarinic receptor agonist that has been reported to cause both PV relaxation23 as well as PV contraction,3,4,13,14 depending 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 cat24 and rat25 and to induce vasorelaxation in precontracted pulmonary arterial rings of the rabbit,26 rat,27 and guinea pig.28 In 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.16 In 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).29 At least two types of VOCCs are present in vascular smooth muscle: “transient” (T-type) channels and “long-lasting” (L-type) channels.30,31 In most vascular smooth muscle cells, L channels are more numerous and probably are the most important route of calcium influx.32,33 T channels are relatively resistant to inhibition by dihydropyridine antagonists (nifedipine and its derivatives), whereas L channels are highly sensitive to dihydropyridine antagonists.34,35 Because 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 muscle36,37 as well as canine airway smooth muscle.38 In addition, ketamine has been reported to decrease IP3formation in response to norepinephrine in neonatal rat cardiomyocytes.39 In 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.40 When activated, PKC may directly or indirectly inhibit myosin light chain phosphatase,40,41 thereby increasing regulatory myosin light chain phosphorylation and force for a given [Ca2+]i.42 Agonist-induced activation of the ROK signaling pathway can also increase myofilament Ca2+sensitivity by inhibiting myosin light chain phosphatase.43 However, only two studies have investigated agonist-induced changes in myofilament Ca2+sensitivity in PVs.5,7 PKC was shown to mediate sustained contraction in response to endothelin,7 whereas norepinephrine and thromboxane A2increased Ca2+sensitivity via  the ROK and tyrosine kinase signaling pathways.5 In 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.44 Ketamine 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.  45 reported that ketamine had no effect on myofilament Ca2+sensitivity in either membrane-permeabilized or membrane-intact rat mesenteric resistance arterial strips. Hanazaki et al.  46 reported 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,47 which likely explains why ketamine had no effect on Ca2+sensitivity in the presence of acetylcholine in their previous study.46 We 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,48 whereas the opposite effect was reported in critically ill and acutely traumatized patients.49 We 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,1 our 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.50 Ketamine 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.
References
Barnes PJ, Liu SF: Regulation of pulmonary vascular tone. Pharmacol Rev 1995; 47:87–131Barnes, PJ Liu, SF
Joiner PD, Kadowitz PJ, Davis LB, Hyman AL: Contractile responses of canine isolated pulmonary lobar arteries and veins to norepinephrine, serotonin, and tyramine. Can J Physiol Pharmacol 1975; 53:830–8Joiner, PD Kadowitz, PJ Davis, LB Hyman, AL
Shi W, Eidelman DH, Michel RP: Differential relaxant responses of pulmonary arteries and veins in lung explants of guinea pigs. J Appl Physiol 1997; 83:1476–81Shi, W Eidelman, DH Michel, RP
Toga H, Bansal V, Raj JU: Differential responses of ovine intrapulmonary arteries and veins to acetylcholine. Respir Physiol 1996; 104:197–204Toga, H Bansal, V Raj, JU
Janssen LJ, Lu-Chao H, Netherton S: Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho kinase. Am J Physiol Lung Cell Mol Physiol 2001; 280:L666–74Janssen, LJ Lu-Chao, H Netherton, S
Michelakis ED, Weir EK, Wu X, Nsair A, Waite R, Hashimoto K, Puttagunta L, Knaus HG, Archer SL: Potassium channels regulate tone in rat pulmonary veins. Am J Physiol Lung Cell Mol Physiol 2001; 280:L1138–47Michelakis, ED Weir, EK Wu, X Nsair, A Waite, R Hashimoto, K Puttagunta, L Knaus, HG Archer, SL
Steffan M, Russell JA: Signal transduction in endothelin-induced contraction of rabbit pulmonary vein. Pulm Pharmacol 1990; 3:1–7Steffan, M Russell, JA
Sudjarwo SA, Hori M, Tanaka T, Matsuda Y, Karaki H: Coupling of the endothelin ETAand ETBreceptors to Ca2+mobilization and Ca2+sensitization in vascular smooth muscle. Eur J Pharmacol 1995; 289:197–204Sudjarwo, SA Hori, M Tanaka, T Matsuda, Y Karaki, H
Jing M, Ledvina MA, Bina S, Hart JL, Muldoon SM: Effects of halogenated and non-halogenated anesthetics on diaspirin cross-linked hemoglobin induced contractions of porcine pulmonary veins. Artif Cells Blood Substit Immobil Biotechnol 1995; 23:487–94Jing, M Ledvina, MA Bina, S Hart, JL Muldoon, SM
Chand N, Altura BM: Acetylcholine and bradykinin relax intrapulmonary arteries by acting on endothelial cells: Role in lung vascular diseases. Science 1981; 213:1376–9Chand, N Altura, BM
Hyman AL, Kadowitz PJ: Tone-dependent responses to acetylcholine in the feline pulmonary vascular bed. J Appl Physiol 1988; 64:2002–9Hyman, AL Kadowitz, PJ
Horibe M, Ogawa K, Sohn JT, Murray PA: Propofol attenuates acetylcholine-induced pulmonary vasorelaxation: Role of nitric oxide and endothelium-derived hyperpolarizing factors. Anesthesiology 2000; 93:447–55Horibe, M Ogawa, K Sohn, JT Murray, PA
Steinhorn RH, Morin FC III, Gugino SF, Giese EC, Russell JA: Developmental differences in endothelium-dependent responses in isolated ovine pulmonary arteries and veins. Am J Physiol 1993; 264:H2162–7Steinhorn, RH Morin, FC Gugino, SF Giese, EC Russell, JA
Gruetter CA, Lemke SM: Comparison of endothelium-dependent relaxation in bovine intrapulmonary artery and vein by acetylcholine and A23187. J Pharmacol Exp Ther 1986; 238:1055–62Gruetter, CA Lemke, SM
Ding X, Murray PA: Regulation of pulmonary venous tone in response to muscarinic receptor activation. Am J Physiol Lung Cell Mol Physiol 2005; 288:L131–40Ding, X Murray, PA
Ogawa K, Tanaka S, Murray PA: Inhibitory effects of etomidate and ketamine on endothelium-dependent relaxation in canine pulmonary artery. Anesthesiology 2001; 94:668–77Ogawa, K Tanaka, S Murray, PA
Feletou M, Girard V, Canet E: Different involvement of nitric oxide in endothelium-dependent relaxation of porcine pulmonary artery and vein: Influence of hypoxia. J Cardiovasc Pharmacol 1995; 25:665–73Feletou, M Girard, V Canet, E
Greenberg S, Xie J, Wang Y, Cai B, Kolls J, Nelson S, Hyman A, Summer WR, Lippton H: Tumor necrosis factor-alpha inhibits endothelium-dependent relaxation. J Appl Physiol 1993; 74:2394–403Greenberg, S Xie, J Wang, Y Cai, B Kolls, J Nelson, S Hyman, A Summer, WR Lippton, H
Aubin JE: Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem 1979; 27:36–43Aubin, JE
Thomas AP, Delaville F: The use of fluorescent indicators for measurements of cytosolic-free calcium concentration in cell populations and single cells, Cellular Calcium: A Practical Approach. Edited by McCormack JG, Cobbold PH. New York, Oxford, 1991, pp 1–54Thomas, AP Delaville, F McCormack JG, Cobbold PH New York Oxford
Haissaguerre M, Jais P, Shah DC, Takahashi A, Hocini M, Quiniou G, Garrigue S, Le Mouroux A, Le Metayer P, Clementy J: Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N Engl J Med 1998; 339:659–66Haissaguerre, M Jais, P Shah, DC Takahashi, A Hocini, M Quiniou, G Garrigue, S Le Mouroux, A Le Metayer, P Clementy, J
Wallick DW, Martin PJ: Separate parasympathetic control of heart rate and atrioventricular conduction of dogs. Am J Physiol 1990; 259:H536–42Wallick, DW Martin, PJ
Gao Y, Zhou H, Raj JU: Endothelium-derived nitric oxide plays a larger role in pulmonary veins than in arteries of newborn lambs. Circ Res 1995; 76:559–65Gao, Y Zhou, H Raj, JU
Kaye AD, Banister RE, Fox CJ, Ibrahim IN, Nossaman BD: Analysis of ketamine responses in the pulmonary vascular bed of the cat. Crit Care Med 2000; 28:1077–82Kaye, AD Banister, RE Fox, CJ Ibrahim, IN Nossaman, BD
Kaye AD, Banister RE, Anwar M, Feng CJ, Kadowitz PJ, Nossaman BD: Pulmonary vasodilation by ketamine is mediated in part by L-type calcium channels. Anesth Analg 1998; 87:956–62Kaye, AD Banister, RE Anwar, M Feng, CJ Kadowitz, PJ Nossaman, BD
Lee TS, Hou X: Vasoactive effects of ketamine on isolated rabbit pulmonary arteries. Chest 1995; 107:1152–5Lee, TS Hou, X
Maruyama K, Maruyama J, Yokochi A, Muneyuki M, Miyasaka K: Vasodilatory effects of ketamine on pulmonary arteries in rats with chronic hypoxic pulmonary hypertension. Anesth Analg 1995; 80:786–92Maruyama, K Maruyama, J Yokochi, A Muneyuki, M Miyasaka, K
Abdalla SS, Laravuso RB, Will JA: Mechanisms of the inhibitory effect of ketamine on guinea pig isolated main pulmonary artery. Anesth Analg 1994; 78:17–22Abdalla, SS Laravuso, RB Will, JA
Godfraind T, Govoni S: Recent advances in the pharmacology of Ca2+and K+channels. Trends Pharmacol Sci 1995; 16:1–4Godfraind, T Govoni, S
Pelzer D, Pelzer S, McDonald TF: Properties and regulation of calcium channels in muscle cells. Rev Physiol Biochem Pharmacol 1990; 114:107–207Pelzer, D Pelzer, S McDonald, TF
Tsien RW, Ellinor PT, Horne WA: Molecular diversity of voltage-dependent Ca2+channels. Trends Pharmacol Sci 1991; 12:349–54Tsien, RW Ellinor, PT Horne, WA
Kuga T, Sadoshima J, Tomoike H, Kanaide H, Akaike N, Nakamura M: Actions of Ca2+antagonists on two types of Ca2+channels in rat aorta smooth muscle cells in primary culture. Circ Res 1990; 67:469–80Kuga, T Sadoshima, J Tomoike, H Kanaide, H Akaike, N Nakamura, M
Orallo F, Salaices M, Alonso MJ, Marin J, Sanchez-Garcia P: Effects of several calcium channels modulators on the [3H]noradrenaline release and 45Ca influx in the rat vas deferens. Gen Pharmacol 1992; 23:257–62Orallo, F Salaices, M Alonso, MJ Marin, J Sanchez-Garcia, P
Godfraind T: Calcium antagonists and vasodilatation. Pharmacol Ther 1994; 64:37–75Godfraind, T
Orallo F: Regulation of cytosolic calcium levels in vascular smooth muscle. Pharmacol Ther 1996; 69:153–71Orallo, F
Baum VC, Tecson ME: Ketamine inhibits transsarcolemmal calcium entry in guinea pig myocardium: Direct evidence by single cell voltage clamp. Anesth Analg 1991; 73:804–7Baum, VC Tecson, ME
Rusy BF, Amuzu JK, Bosscher HA, Redon D, Komai H: Negative inotropic effect of ketamine in rabbit ventricular muscle. Anesth Analg 1990; 71:275–8Rusy, BF Amuzu, JK Bosscher, HA Redon, D Komai, H
Pabelick CM, Jones KA, Street K, Lorenz RR, Warner DO: Calcium concentration-dependent mechanisms through which ketamine relaxes canine airway smooth muscle. Anesthesiology 1997; 86:1104–11Pabelick, CM Jones, KA Street, K Lorenz, RR Warner, DO
Kudoh A, Kudoh E, Katagai H, Takazawa T: Ketamine suppresses norepinephrine-induced inositol 1,4,5-trisphosphate formation via pathways involving protein kinase C. Anesth Analg 2002; 94:552–7Kudoh, A Kudoh, E Katagai, H Takazawa, T
Torphy TJ, Hay DWP: Biochemical regulation of airway smooth-muscle tone: An overview, Airway Smooth Muscle: Modulation of Receptors and Response. Edited by Agrawal DK, Townley RG. Boca Raton, CRC Press Publishers, 1990, pp 39–68Torphy, TJ Hay, DWP Agrawal DK, Townley RG Boca Raton CRC Press Publishers
Somlyo AP, Somlyo AV: Signal transduction and regulation in smooth muscle. Nature 1994; 372:231–6Somlyo, AP Somlyo, AV
Kitazawa T, Gaylinn BD, Denney GH, Somlyo AP: G-protein-mediated Ca2+sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 1991; 266:1708–15Kitazawa, T Gaylinn, BD Denney, GH Somlyo, AP
Kitazawa T, Eto M, Woodsome TP, Brautigan DL: Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 2000; 275:9897–900Kitazawa, T Eto, M Woodsome, TP Brautigan, DL
Yoshimura Y, Yamaguchi O: Calcium independent contraction of bladder smooth muscle. Int J Urol 1997; 4:62–7Yoshimura, Y Yamaguchi, O
Akata T, Izumi K, Nakashima M: Mechanisms of direct inhibitory action of ketamine on vascular smooth muscle in mesenteric resistance arteries. Anesthesiology 2001; 95:452–62Akata, T Izumi, K Nakashima, M
Hanazaki M, Jones KA, Warner DO: Effects of intravenous anesthetics on Ca2+sensitivity in canine tracheal smooth muscle. Anesthesiology 2000; 92:133–9Hanazaki, M Jones, KA Warner, DO
Bremerich DH, Warner DO, Lorenz RR, Shumway R, Jones KA: Role of protein kinase C in calcium sensitization during muscarinic stimulation in airway smooth muscle. Am J Physiol 1997; 273:L775–81Bremerich, DH Warner, DO Lorenz, RR Shumway, R Jones, KA
Gassner S, Cohen M, Aygen M, Levy E, Ventura E, Shadhdi J: The effect of ketamine on pulmonary artery pressure: An experimental and clinical study. Anaesthesia 1974; 29:141–6Gassner, S Cohen, M Aygen, M Levy, E Ventura, E Shadhdi, J
Waxman K, Shoemaker WC, Lippmann M: Cardiovascular effects of anesthetic induction with ketamine. Anesth Analg 1980; 59:355–8Waxman, K Shoemaker, WC Lippmann, M
Domino EF, Zsigmond EK, Domino LE, Domino KE, Kothary SP, Domino SE: Plasma levels of ketamine and two of its metabolites in surgical patients using a gas chromatographic mass fragmentographic assay. Anesth Analg 1982; 61:87–92Domino, EF Zsigmond, EK Domino, LE Domino, KE Kothary, SP Domino, SE
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
Fig. 1. (  A  ) Effect of ketamine (10−5m to 10−3m) on acetylcholine (ACh) contraction in isolated canine endothelium-intact (E+) pulmonary vein (PV) rings. (  B  ) Effect of ketamine on acetylcholine contraction in isolated canine endothelium-denuded (E−) PV rings. Ketamine (10−5m) had no effect (E+:  P  = 0.774; E−:  P  = 0.661), whereas ketamine (10−4m and 10−3m) attenuated acetylcholine contraction in E+ (  P  = 0.001 and  P  = 0.001, respectively) and E− PV rings (  P  < 0.001 and  P  < 0.001, respectively). n = 7.  Error bars  represent SDs in all figures. 
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Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
Fig. 2. Effect of nitric oxide synthase (NOS) inhibition (  N  -nitro-l-arginine methylester [l-NAME; 10−4m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-intact pulmonary vein rings. Acetylcholine contraction was potentiated by nitric oxide synthase inhibition (  P  < 0.001), but the ketamine-induced attenuation in acetylcholine contraction was still observed after NOS inhibition (  P  = 0.002). n = 6. 
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Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
Fig. 3. (  A  ) Effect of voltage-operated Ca2+channel inhibition (nifedipine [10−5m]), alone and in combination with ketamine, on acetylcholine (ACh) contraction in endothelium-denuded pulmonary vein rings. (  B  ) Effect of inositol-1,4,5-trisphosphate (IP3)–mediated Ca2+release inhibition (2-aminoethoxydiphenylborate [2-APB; 10−4m]), alone and in combination with ketamine, on acetylcholine contraction in endothelium-denuded pulmonary vein rings. Acetylcholine contraction was attenuated by inhibition of voltage-operated Ca2+channels and IP3-mediated Ca2+release (  P  < 0.001 and  P  < 0.001, respectively), but the ketamine-induced attenuation in acetylcholine contraction was still observed after inhibition of voltage-operated Ca2+channels (  P  < 0.001) or IP3-mediated Ca2+release (  P  = 0.021). n = 6. 
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Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
Fig. 4. (  A  ) Effect of ketamine on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of ketamine on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of ketamine on the pulmonary vein [Ca2+]i–tension relation. Ketamine had no effect on increases in tension (  P  = 0.875), [Ca2+]i(  P  = 0.869), or the [Ca2+]i–tension relation (  P  = 0.892). n = 6. 
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Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 5. (  A  ) Effect of acetylcholine (ACh; 10−6m), alone and in combination with ketamine, on extracellular Ca2+-induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of acetylcholine, alone and in combination with ketamine, on extracellular Ca2+-induced increases in intracellular Ca2+concentration ([Ca2+]i) in endothelium-denuded pulmonary vein strips. (  C  ) Effect of acetylcholine, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation. Acetylcholine increased tension (  P  = 0.004), had no effect on [Ca2+]i(  P  = 0.836), and caused a leftward shift (  P  = 0.004) in the [Ca2+]i–tension relation. Ketamine attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.836), and caused a rightward shift (  P  = 0.038) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 6. (  A  ) Effect of rho-kinase inhibition (Y27632 [10−6m]), alone and in combination with ketamine, on tension in acetylcholine (ACh)–pretreated endothelium-denuded pulmonary vein strips. (  B  ) Effect of Y27632, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of Y27632, alone and in combination with ketamine, on the pulmonary veins [Ca2+]i–tension relation in acetylcholine-pretreated strips. Y27632 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of Y27632, ketamine further attenuated (  P  = 0.034) the acetylcholine-induced increases in tension, had no effect on [Ca2+]i(  P  = 0.843), and caused a rightward shift (  P  = 0.039) in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
Fig. 7. (  A  ) Effect of protein kinase C inhibition (bisindolylmaleimide I [BIS1; 3 × 10−6m]), alone and in combination with ketamine, on acetylcholine (ACh)–induced increases in tension in endothelium-denuded pulmonary vein strips. (  B  ) Effect of BIS1, alone and in combination with ketamine, on intracellular Ca2+concentration ([Ca2+]i) in acetylcholine-pretreated endothelium-denuded pulmonary vein strips. (  C  ) Effect of BIS1, alone and in combination with ketamine, on the pulmonary vein [Ca2+]i–tension relation in acetylcholine-pretreated strips. BIS1 decreased acetylcholine-induced increases in tension (  P  < 0.001), had no effect on [Ca2+]i(  P  = 0.193), and caused a rightward shift (  P  < 0.001) in the [Ca2+]i–tension relation. In the presence of BIS1, the ketamine-induced attenuation of the acetylcholine-induced increases in tension is abolished (  P  = 0.607), with no effect on [Ca2+]i(  P  = 0.193). Therefore, protein kinase C inhibition abolished (  P  = 0.798) the ketamine-induced change in the [Ca2+]i–tension relation in acetylcholine-pretreated pulmonary vein strips. n = 6. 
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