Propofol Increases Pulmonary Artery Smooth Muscle Myofilament Calcium Sensitivity: Role of Protein Kinase C

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

Healthy male mongrel dogs weighing 24–32 kg were anesthetized with pentobarbital sodium (30 mg/kg, intravenous) and fentanyl citrate (15 μg/kg, intravenous). After tracheal intubation, the lungs were mechanically ventilated. A catheter was placed in the right femoral artery, and the dogs were killed 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 pulmonary arteries (2–4 mm ID) were dissected carefully and immersed in cold modified Krebs-Ringer's bicarbonate (KRB) solution. The arteries were cleaned of connective tissue and cut into strips (2 × 8 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 acetylcholine (10⁻⁶M).

Pulmonary arterial strips without endothelium were loaded with 5 × 10⁻⁶m acetoxylmethyl ester of fura-2 (fura-2/AM) solution, as described previously. 3A noncytotoxic detergent, 0.05% Cremophor EL (Sigma Chemical, St. Louis, MO), was added to solubilize the fura-2/AM in the solution. After fura-2 loading, the arterial strips were washed with KRB buffer to remove uncleaved fura-2/AM and 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% air and 5% CO²(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 4.0 g, which was determined in preliminary studies to be optimal for achieving a maximum contractile response to 30 mm KCl. 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 concentrations 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 measured in all experiments, and the signals were observed to change in opposite directions in response to the various interventions. Because each PASM strip served as its own control, background fluorescence was assumed to be constant and was not subtracted from the calculated 340/380 ratio. 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 collected with a software package from Photon Technology International.

We measured tension and [Ca²⁺]ⁱsimultaneously to investigate how propofol alters the relation between tension and [Ca²⁺]ⁱin pulmonary artery smooth muscle. In protocol 1, time control experiments were performed to assess the reproducibility of KCl-induced increases in [Ca²⁺]ⁱand tension. The PASM strips (n = 6) were treated with 30 mm KCl. After changes in [Ca²⁺]ⁱand tension had reached new steady state values (10–15 min), the strips were washed with fresh KRB solution and [Ca²⁺]ⁱand tension returned to baseline. After a return to baseline, a period of 10–15 min was allowed before the second application of 30 mm KCl. This same procedure was followed for the third application of 30 mm KCl.

In protocol 2, we tested the hypothesis that the Ca²⁺channel antagonist, verapamil, and the intravenous anesthetic propofol would have differential effects on KCl-induced changes in [Ca²⁺]ⁱand tension. Once again the experimental design was similar to protocol 1. After the first application of KCl, the PASM strips (n = 6, each) were pretreated with either propofol (10 μm) or verapamil (0.1 μm) for 10 min. This was followed by the second application of 30 mm KCl. The strips were then washed with fresh KRB buffer. After a return to baseline values, the strips were pretreated with either propofol (100 μm) or verapamil (0.3 μm) for 10 min. This was followed by the third application of 30 mm KCl. In additional PASM strips (n = 5), a similar procedure was followed to assess the effects of higher concentrations of verapamil (1 and 10 μm) on KCl-induced increases in [Ca²⁺]ⁱand tension.

In protocol 3, we investigated the effects of propofol on the [Ca²⁺]ⁱ-tension relation by increasing the extracellular Ca²⁺concentration. In each PASM strip (n = 5), the response to 30 mm KCl was assessed first. After washout, the strips were treated with a Ca²⁺-free buffer containing 2 mm EGTA for 10 min. This solution was replaced with a Ca²⁺-free solution that did not contain EGTA. After 10 min, this solution was replaced with a Ca²⁺-free solution containing 30 mm KCl. Finally, after 10 min, the extracellular Ca²⁺concentration was increased in control and propofol-pretreated (100 μm) strips in an incremental fashion from 0 mm to 0.25, 0.5, 1, and 3 mm. In protocols 1–3, changes in tension and [Ca²⁺]ⁱare expressed as a percentage of the response to the first application of 30 mm KCl.

In protocol 4, we eliminated the possible inhibitory effect of propofol on increases in [Ca²⁺]ⁱin response to KCl-induced depolarization, 4which could mask a propofol-induced increase in myofilament Ca²⁺sensitivity. To do this, we performed experiments where [Ca²⁺]ⁱwas increased using the Ca²⁺ionophore ionomycin (0.1 μm). First, increases in tension and [Ca²⁺]ⁱin response to 60 mm KCl were obtained. We used 60 mm KCl as our reference response in this protocol because in preliminary experiments we observed that ionomycin increased [Ca²⁺]ⁱto approximately 50% of the value induced by 60 mm KCl. The strips were then washed with fresh KRB solution. After tension and [Ca²⁺]ⁱhad returned to baseline, ionomycin (0.1 μm) was administered. This concentration of ionomycin had essentially no effect on tension, whereas it increased [Ca²⁺]ⁱto approximately 50% of the value induced by 60 mm KCl. In this setting, propofol (1, 10, 100 μm) was administered cumulatively to the perfusion solution. The same procedure was repeated in strips pretreated with bisindolylmaleimide I (BIS, 1 μm), a specific PKC inhibitor, 5or Gö 6976 (10 μm), a selective inhibitor of Ca²⁺-dependent PKC isoforms. 6We also investigated the effects of phorbol 12-myristate 13-acetate (PMA, 1 μm), a specific PKC activator, on tension and [Ca²⁺]ⁱin ionomycin-treated strips in the presence and absence of BIS. To determine whether an increase in [Ca²⁺]ⁱis required for propofol-induced changes in tension, propofol (1, 10, 100 μm) was administered to PASM strips (n = 3) in the absence of extracellular Ca²⁺(Ca²⁺-free solution containing 2 mm EGTA) and after pretreatment with the IP³-receptor antagonist 2-aminoethoxydiphenyl borate (2-APB, 100 μm, Calbiochem, La Jolla, CA), to inhibit propofol-induced changes in Ca²⁺release from the sarcoplasmic reticulum. 4In protocol 4, changes in tension and [Ca²⁺]ⁱare expressed as a percentage of the response to 60 mm KCl.

The KRB solution had the following composition: 118.3 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO⁴, 1.2 mm KH²PO⁴, 2.5 mm CaCl², 25 mm NaHCO³, 0.016 mm Ca-EDTA, and 11.1 mm glucose, gassed with 95% air and 5% CO²at 37°C, pH 7.4. The KCl solutions were prepared in an isotonic fashion by replacing NaCl with equimolar KCl. The following chemicals were used: fura-2/AM (Texas Fluorescence Labs, Austin, TX), acetylcholine chloride, ionomycin, BIS, PMA, Cremophor EL, Gö 6976, and 2-APB. The BIS, Gö 6976, and ionomycin were dissolved in dimethyl sulfoxide and diluted with distilled water. The final concentration of dimethyl sulfoxide in the organ bath was less than 0.1% (vol/vol). In control experiments, none of the agents or solutions used in this study caused significant shifts in the fluorescence signals in fura-2 nonloaded PASM strips, suggesting that changes in fluorescence of endogenous substances are negligible.

All values are expressed as mean ± SEM. Statistical analysis used analysis of variance, Student paired t  test, and the Bonferroni test for multiple comparisons. The peak tension-[Ca²⁺]ⁱrelations were compared using linear regression analysis. A P  value of less than 0.05 was considered statistically significant. In all experiments, n equals the number of dogs from which the PASM strips were obtained. For each protocol, multiple strips from the same dog were averaged, so that all dogs were weighted equally in the analysis.

Figure 1illustrates changes in tension and the 340, 380, and 340/380 fura-2 fluorescence signals in a PASM strip in response to three sequential applications of 30 mm KCl. Increases in [Ca²⁺]ⁱand tension in response to the second and third application of 30 mm KCl are summarized as a percentage of the first KCl response. Changes in tension (108 ± 5 and 99 ± 4%) and [Ca²⁺]ⁱ(96 ± 2 and 90 ± 3%) in response to sequential treatments of 30 mm KCl were highly reproducible.

Figure 2illustrates the effects of verapamil and propofol on original recordings of changes in [Ca²⁺]ⁱand tension induced by 30 mm KCl. The KCl caused a monotonic increase in tension that was maintained at a peak value as well as a monotonic increase in [Ca²⁺]ⁱthat slightly decreased from a peak value. Inhibition of L-type Ca²⁺channels with verapamil (0.1 and 0.3 μm) caused dose-dependent decreases in [Ca²⁺]ⁱand tension of equal magnitude. Propofol (10 and 100 μm) also caused dose-dependent decreases in [Ca²⁺]ⁱ. However, tension was relatively well preserved in the presence of propofol. To further contrast the effects of verapamil and propofol on KCl-induced increases in [Ca²⁺]ⁱand tension, the data presented in figure 2are plotted as phase-plane loops showing the continuous [Ca²⁺]ⁱ-tension relation (fig. 3). The dashed arrows connect the points where the maximum tension and the corresponding [Ca²⁺]ⁱwere achieved. Verapamil attenuated both peak tension and [Ca²⁺]ⁱto a similar extent, as indicated by the slope of the arrow approximately passing through unity. In contrast, propofol caused an upward shift in the relation between peak tension and [Ca²⁺]ⁱcompared with verapamil. Propofol significantly decreased the slope of the peak tension-[Ca²⁺]ⁱrelation compared with verapamil (0.64 ± 0.08 vs.  1.07 ± 0.08, respectively, P < 0.05). When higher concentrations of verapamil (1 and 10 μm) were used to further reduce KCl-induced increases in tension and [Ca²⁺]ⁱ, the slope of the peak tension-[Ca²⁺]ⁱrelation (1.17 ± 0.10) was similar to that observed with the lower concentrations of verapamil and significantly higher (P < 0.05) than that observed with propofol. Moreover, PKC inhibition with BIS completely reversed the propofol-induced decrease in the slope of the peak tension-[Ca²⁺]ⁱrelation in response to KCl depolarization (BIS plus propofol, 100 μm, slope = 1.26 ± 0.10). These results suggest that propofol increases myofilament Ca²⁺sensitivity in PASM via  a PKC-sensitive pathway.

To more directly assess the effects of propofol on myofilament Ca²⁺sensitivity, control and propofol-pretreated PASM strips bathed in a Ca²⁺-free buffer containing 30 mm KCl were exposed to incremental increases in extracellular Ca²⁺concentration. As summarized in figure 4, increasing extracellular Ca²⁺concentration resulted in virtually identical increases in [Ca²⁺]ⁱin control and propofol-pretreated strips, whereas concomitant increases in tension were greater (*P < 0.05) in propofol-pretreated strips compared with control. This resulted in a shift in the [Ca²⁺]ⁱ-tension relation, such that for a given value of [Ca²⁺]ⁱ, tension was greater in the propofol-pretreated strips compared with the control.

Figure 5illustrates original recordings of changes in [Ca²⁺]ⁱand tension induced by propofol in the presence or absence of the specific PKC inhibitor, BIS. To eliminate the inhibitory effect of propofol on increases in [Ca²⁺]ⁱin response to KCl-induced depolarization, which could mask a propofol-induced increase in myofilament Ca²⁺sensitivity, the PASM strips were pretreated with the Ca²⁺ionophore, ionomycin (0.1 μm), to increase [Ca²⁺]ⁱ. Ionomycin had essentially no effect on tension, whereas it increased [Ca²⁺]ⁱto approximately 50% of the value induced by 60 mm KCl (fig. 5). During these conditions, propofol caused a dose-dependent increase in tension but had little effect on [Ca²⁺]ⁱ. Pretreatment with BIS before ionomycin administration attenuated the increases in tension in response to propofol. Summarized data are shown in figure 6. Propofol significantly increased tension, whereas [Ca²⁺]ⁱwas slightly decreased. Pretreatment with BIS attenuated the propofol-induced changes in tension and [Ca²⁺]ⁱ.

Figure 7summarizes the effects of the PKC activator PMA on [Ca²⁺]ⁱand tension in ionomycin-pretreated strips in the presence and absence of BIS. As observed with propofol, PMA increased tension, whereas [Ca²⁺]ⁱwas slightly decreased. Pretreatment with BIS abolished the PMA-induced changes in tension and [Ca²⁺]ⁱ. These results indicate that increases in myofilament Ca²⁺sensitivity in response to PMA involve the PKC signaling pathway.

As summarized in figure 8, pretreatment with Gö 6976, a selective inhibitor of Ca²⁺-dependent PKC isoforms, also attenuated the propofol-induced increase in tension and the decrease in [Ca²⁺]ⁱin ionomycin-pretreated strips, though this effect was only apparent at the highest concentration of propofol. To determine whether an increase in [Ca²⁺]ⁱis required for the propofol-induced increase in tension, propofol (1, 10, and 100 μm) was administered to the PASM strips in the absence of extracellular Ca²⁺and after pretreatment with the IP³receptor antagonist 2-APB to inhibit Ca²⁺release from the sarcoplasmic reticulum. During these conditions, propofol had no effect on tension or [Ca²⁺]ⁱ.

This is the first study to assess the effects of propofol on myofilament Ca²⁺sensitivity in PASM. Our results demonstrate that propofol increases myofilament Ca²⁺sensitivity. Moreover, this effect is attenuated by a specific inhibitor of PKC and an inhibitor of Ca²⁺-dependent PKC isoforms. These results suggest that the propofol-induced increase in myofilament Ca²⁺sensitivity involves the PKC signaling pathway.

The results from protocol 1 demonstrated that triplicate responses to KCl cause reproducible and consistent increases in [Ca²⁺]ⁱand tension. Our next goal was to determine whether propofol alters the relation between KCl-induced changes in tension and [Ca²⁺]ⁱin PASM. The KCl-induced increases in tension are mediated by Ca²⁺influx through L-type, voltage-gated Ca²⁺channels, which results in an increase in [Ca²⁺]ⁱ, phosphorylation of myosin light chain, and tension development. Thus, the L-type Ca²⁺channel blocker verapamil would be expected to inhibit KCl-induced increases in tension and [Ca²⁺]ⁱto a similar extent, as was reported previously in isolated guinea pig aortae. 7This assumes that verapamil has no effect on myofilament Ca²⁺sensitivity, which was reported in canine coronary arteries. 8In contrast to verapamil, propofol attenuated KCl-induced increases in [Ca²⁺]ⁱto a greater extent than concomitant increases in tension. This was reflected by an upward shift in the peak tension-[Ca²⁺]ⁱrelation, suggesting an increase in myofilament Ca²⁺sensitivity. It is important to note that this method to assess the effects of propofol on myofilament Ca²⁺sensitivity has limitations (e.g. , unknown effects of propofol on neurotransmitter release) and does not alone provide direct proof that propofol increases myofilament Ca²⁺sensitivity. To more directly assess this possibility, we investigated the effects of propofol in PASM strips perfused with a Ca²⁺-free solution containing 30 mm KCl (protocol 3). During these conditions, increasing extracellular Ca²⁺concentration in an incremental fashion caused virtually identical increases in [Ca²⁺]ⁱin control and propofol-pretreated strips, whereas concomitant increases in tension were greater in propofol-pretreated strips. Thus, for a given value of [Ca²⁺]ⁱ, tension was greater during propofol compared with control; that is, propofol increased myofilament Ca²⁺sensitivity. Taken together, our results suggest that propofol has at least two opposing effects on KCl-induced contraction. Propofol has been shown to inhibit L-type, voltage-gated Ca²⁺channels in systemic vascular smooth muscle, 4,9tracheal smooth muscle cells, 10and myocardial cells. 11Thus, it is likely that the inhibitory effect of propofol on KCl-induced increases in tension and [Ca²⁺]ⁱin PASM is due to inhibition of L-type, voltage-gated Ca²⁺channels. However, this effect is partially offset by a concomitant propofol-induced increase in PASM myofilament Ca²⁺sensitivity.

The effects of propofol on myofilament Ca²⁺sensitivity may depend on the species and/or the tissue type being investigated. Propofol had no effect on myofilament Ca²⁺sensitivity in the absence or presence of muscarinic receptor stimulation in β-escin-permeabilized canine tracheal smooth muscle, even at supraclinical concentrations. 12Propofol was reported to decrease myofilament Ca²⁺sensitivity in rat ventricular myocytes at supraclinical concentrations. 13In contrast, Nakae et al.  14reported that clinically relevant concentrations of propofol increased myofilament Ca²⁺sensitivity in isolated guinea pig hearts. Our laboratory 15has reported that propofol increases myofilament Ca²⁺sensitivity in rat cardiomyocytes. Recently, we 16demonstrated in cardiomyocytes that propofol increases the sensitivity of myofibrillar actomyosin adenosine triphosphatase to Ca²⁺(i.e. , increases myofilament Ca²⁺sensitivity), at least in part by increasing intracellular pH via  a PKC-dependent activation of Na⁺-H⁺exchange. In the present study, propofol caused an upward shift in the PASM peak tension-[Ca²⁺]ⁱrelation compared with verapamil-induced changes and a shift in the [Ca²⁺]ⁱ-tension relation when extracellular Ca²⁺concentration was increased incrementally from a Ca²⁺-free condition. Moreover, in ionomycin-pretreated PASM strips, propofol caused a dose-dependent increase in tension without a concomitant increase in [Ca²⁺]ⁱ. These results clearly indicate that propofol increases myofilament Ca²⁺sensitivity in PASM.

Based on our previous results in cardiomyocytes, 16we next tested the hypothesis that propofol increases PASM myofilament Ca²⁺sensitivity via  activation of the PKC signaling pathway. In the ionomycin-pretreated PASM strips, the propofol-induced increases in tension and decreases in [Ca²⁺]ⁱwere attenuated by BIS, a specific inhibitor of PKC. Moreover, the PKC activator PMA mimicked the effects of propofol in the ionomycin-pretreated PASM strips; that is, PMA also increased tension and decreased [Ca²⁺]ⁱ. These PMA-induced changes were abolished by BIS. Taken together, these results suggest that the propofol-induced increase in myofilament Ca²⁺sensitivity and the concomitant decrease in [Ca²⁺]ⁱinvolve PKC activation.

The primary mechanisms of contraction in vascular smooth muscle involve Ca²⁺/calmodulin-dependent, MLCK-dependent phosphorylation of LC20, 17,18and myofilament Ca²⁺sensitization. 19The PKC has been implicated in the signaling pathway that regulates myofilament Ca²⁺sensitivity. 20Some reports 21,22indicate that PKC-mediated contraction is Ca²⁺dependent, 23–25whereas other studies suggest that it is Ca²⁺independent. We recently identified six isoforms of PKC in canine PASM cells, 26representing the classic, novel, and atypical PKC isoform groups. Two of the isoforms (PKCα and PKCδ) underwent translocation in response to the PKC activator dioctanoylglycerol and in response to angiotensin II. PKCα is a member of the classic group A PKC isoforms, which are Ca²⁺dependent. PKCβ and PKCγ are also members of the classic group A PKC isoforms but are not present in PASM. 26PKCδ is a member of the novel group B PKC isoforms, which are Ca²⁺independent. In the present study, selective inhibition of Ca²⁺-dependent PKC isoforms with Gö 6976 attenuated the propofol-induced increases in tension and decreases in [Ca²⁺]ⁱ. Also, Gö 6976 was shown to inhibit the vasoconstrictor responses to angiotensin II 27and hypoxia 28in intact lungs. Moreover, our observation that propofol had no effect on tension during experimental conditions that prevented an increase in [Ca²⁺]ⁱ(i.e. , absence of extracellular Ca²⁺and after IP³-receptor block) underscores the Ca²⁺dependence of the propofol-induced, PKC-mediated increase in tension. Taken together, our results suggest that the propofol-induced increase in tension and decrease in [Ca²⁺]ⁱinvolves activation of PKCα.

The cellular mechanisms by which propofol causes PKC-dependent changes in [Ca²⁺]ⁱand tension have yet to be identified. The effect of propofol on [Ca²⁺]ⁱcould be due to either a PKC-mediated decrease in Ca²⁺influx (e.g. , via  L-type, voltage-gated Ca²⁺channels) or an increase in Ca²⁺extrusion. PKC is known to activate Na⁺-Ca²⁺exchange 29and/or Ca²⁺pumps. 30Recently, we 31demonstrated that propofol inhibits capacitative Ca²⁺entry in PASM cells via  a PKC-dependent pathway. It is possible that the small decreases in [Ca²⁺]ⁱin response to propofol and PMA may be due to inhibition of capacitative Ca²⁺entry via  the PKC signaling pathway. PKC has been postulated to increase myofilament Ca²⁺sensitivity via  several mechanisms, including phosphorylation of thin-filament accessory proteins (e.g. , caldesmon, calponin), inhibition of myosin light chain phosphatase, phosphorylation of myosin light chain, and activation of Na⁺-H⁺exchange, resulting in intracellular alkalinization. 19,32In contrast, PKC was not found to play an important role in norepinephrine-induced increases in myofilament Ca²⁺sensitivity in PASM. 33A possible limitation of that study is that [Ca²⁺]ⁱand tension were not measured simultaneously in the same tissue. 33Moreover, the study by Janssen et al.  33used endothelium-intact pulmonary arterial rings that were not pretreated to inhibit the β-agonist effect of norepin-ephrine. These factors could confound interpretation of norepinephrine-induced changes in tension. Finally, it should be noted that neither BIS nor Gö 6976 abolished the propofol-induced increases in PASM tension. This suggests that other signaling mechanisms that regulate myofilament Ca²⁺sensitivity (e.g. , tyrosine kinases, Rho-kinase) could be involved.

Ionomycin is a Ca²⁺ionophore that increases [Ca²⁺]ⁱ, both via  an increase in sarcolemmal Ca²⁺influx and via  Ca²⁺release from intracellular stores. This latter effect could in turn lead to activation of capacitative Ca²⁺entry. As previously reported, 34we observed that ionomycin induced a smaller contraction than that predicted from the concomitant increase in [Ca²⁺]ⁱ. The precise mechanism underlying this ionomycin-induced dissociation between tension and [Ca²⁺]ⁱis unknown. One possible mechanism is that, because ionomycin can inhibit mitochondrial activity, 35,36decreased adenosine triphosphate production may dissociate contraction from increases in [Ca²⁺]ⁱ. Alternatively, ionomycin itself may decrease myofilament Ca²⁺sensitivity. This possibility needs to be considered when interpreting the effects of propofol in the ionomycin-pretreated PASM strips.

The plasma concentration of propofol in patients during maintenance of general anesthesia was reported to be in the range of 10⁻⁵to 10⁻⁴m. 37Because 97–98% of propofol is bound to plasma proteins, 38the free concentration of propofol is approximately 10⁻⁶to 10⁻⁵m. However, protein binding of propofol in vivo  is unlikely to be instantaneous, so the free drug concentration associated with a bolus injection would be higher than the steady state value. Moreover, it was demonstrated recently that 28% of propofol is taken up by the lung during a single passage through the lung, and most of the propofol that undergoes pulmonary uptake is released back into the circulation by back diffusion. 39This results in a higher concentration of propofol in the pulmonary artery than in the radial artery. 40In this study, propofol concentrations of 10⁻⁵m and higher significantly increased tension without increasing [Ca²⁺]ⁱin ionomycin-treated pulmonary arterial strips. This result indicates that propofol can increase PASM myofilament Ca²⁺sensitivity at clinically relevant concentrations.

In conclusion, propofol has a direct inhibitory effect on PASM contraction that is mediated by a decrease in the availability of [Ca²⁺]ⁱ. Propofol also increases PASM myofilament Ca²⁺sensitivity, and this effect is at least partially mediated by the PKC signaling pathway. Indirect evidence suggests an involvement of PKCα. Future studies will be required to determine whether propofol also increases myofilament Ca²⁺sensitivity during receptor activation.