**3. Calming effect of local anesthetics on neutrophils**

In contrast to general inhalation anesthetics, lidocaine has been found to inhibit protein kinase C in a manner competitive with phosphatidylserine as well as phosphorylation of the 47‐kDa neu‐ trophil cytoplasmic protein [11, 14, 15]. Thus, various stimulation‐coupled responses of neutro‐ phils, such as superoxide generation (**Figure 5**), depolarization of membrane potential (**Figure 6**), and transitional increase in intracellular Ca2+ (**Figure 7**), are suppressed by lidocaine. Other local anesthetics have also been shown to inhibit protein kinase C activity [15] and induce the same effects (**Figure 8**). These findings indicate that local anesthetics have unique calming effects on neutrophils as compared with inhalation anesthetics, which may reduce oxidative stress.

**Figure 5.** Effects of lidocaine on superoxide generation by neutrophils. Neutrophils were obtained from a guinea pig and suspended (1 × 10<sup>6</sup> cells·ml−1) in KRP medium containing 10 mM glucose, 1.5 mM NaN<sup>3</sup> , 1 mM Ca2+, and 25 μM cytochrome c. After pre‐incubation for 3 min at 37°C with various concentrations of lidocaine (0–20 mM), 100 nM FMLP (formyl‐methionyl‐leucyl‐phenylalanine) or 0.5 nM TPA (12‐O‐tetradecanoylphorbol‐13‐acetate) was added. Superoxide generation by neutrophils was analyzed by determining the reduction of cytochrome c, which was measured at 550 nm with a reference wavelength of 540 nm. *Left panel*: Time course of cytochrome c reduction induced by FMLP‐stimulated neutrophils. *Right panel*: Percent inhibition of reduction of cytochrome c in FMLP or TPA‐stimulated neutrophils by lidocaine. These results indicated that lidocaine strongly inhibited superoxide generation in neutrophils stimulated by treatment with TPA or FMLP in a dose‐dependent manner.

Regional Anesthesia: Advantages of Combined Use with General Anesthesia and Useful Tips for Improving... http://dx.doi.org/10.5772/66573 31

**Figure 6.** Effects of lidocaine on membrane potential in neutrophils. We used a cyanine dye method with diS‐C3‐(5). Neutrophils (1 × 10<sup>6</sup> cells·ml−1) were obtained from a guinea pig and suspended in KRP medium containing 10 mM glucose, 1 mM Ca2+, and 5 μM diS‐C3‐(5) at 37°C. After pre‐incubation for 3 min with various concentrations of lidocaine, 100 nM FMLP was added. Changes in the fluorescence intensity of diS‐C3‐(5) were monitored at 670 nm after the dye had been exited at 622 nm. Downward deflections in the trace indicate uptake of diS‐C3‐(5) by the cells corresponding to hyperpolarization of the membrane potential. The inserted curved line graph indicates percent inhibition of fluorescence change by addition of lidocaine. Values were obtained by comparisons of peak values. These results indicated that lidocaine inhibited the membrane depolarization of neutrophils induced by FMLP, which was accompanied by superoxide generation.

**Figure 7.** The effects of lidocaine on intracellular Ca2+ concentration. [Ca2+]i in neutrophils was analyzed by determining the fluorescence intensity of Fura‐2, a fluorescent indicator of intracellular calcium. Neutrophils were obtained from a guinea pig and suspended (1 × 10<sup>6</sup> cells·ml−<sup>1</sup> ) in Ca2+‐free KRP medium, then Fura‐2 was added. Neutrophils were pre‐ incubated for 3 min with various concentrations of lidocaine at 37°C, then incubated with 1 mM Ca2+ for 2 min before addition of 50 nM FMLP. [Ca2+]i was calculated based on the change in fluorescence intensity of Fura‐2. A change in intensity indicated that lidocaine inhibited the increase in intracellular Ca2+ during the course of neutrophil activation.

**Figure 5.** Effects of lidocaine on superoxide generation by neutrophils. Neutrophils were obtained from a guinea pig

cytochrome c. After pre‐incubation for 3 min at 37°C with various concentrations of lidocaine (0–20 mM), 100 nM FMLP (formyl‐methionyl‐leucyl‐phenylalanine) or 0.5 nM TPA (12‐O‐tetradecanoylphorbol‐13‐acetate) was added. Superoxide generation by neutrophils was analyzed by determining the reduction of cytochrome c, which was measured at 550 nm with a reference wavelength of 540 nm. *Left panel*: Time course of cytochrome c reduction induced by FMLP‐stimulated neutrophils. *Right panel*: Percent inhibition of reduction of cytochrome c in FMLP or TPA‐stimulated neutrophils by lidocaine. These results indicated that lidocaine strongly inhibited superoxide generation in neutrophils stimulated by

, 1 mM Ca2+, and 25 μM

cells·ml−1) in KRP medium containing 10 mM glucose, 1.5 mM NaN<sup>3</sup>

and suspended (1 × 10<sup>6</sup>

treatment with TPA or FMLP in a dose‐dependent manner.

**3. Calming effect of local anesthetics on neutrophils**

30 Current Topics in Anesthesiology

In contrast to general inhalation anesthetics, lidocaine has been found to inhibit protein kinase C in a manner competitive with phosphatidylserine as well as phosphorylation of the 47‐kDa neu‐ trophil cytoplasmic protein [11, 14, 15]. Thus, various stimulation‐coupled responses of neutro‐ phils, such as superoxide generation (**Figure 5**), depolarization of membrane potential (**Figure 6**), and transitional increase in intracellular Ca2+ (**Figure 7**), are suppressed by lidocaine. Other local anesthetics have also been shown to inhibit protein kinase C activity [15] and induce the same effects (**Figure 8**). These findings indicate that local anesthetics have unique calming effects on

neutrophils as compared with inhalation anesthetics, which may reduce oxidative stress.

**Figure 8.** Effects of lidocaine and other local anesthetics on PKC activity in rat brain. PKC was obtained from a rat brain and purified using a method previously described [10]. Enzyme activity was assessed by measuring the incorporation of 32P from [γ‐<sup>32</sup>P]ATP into H1 histone (type IIIs) at 30°C for 3 min. The medium contained 20 mM Tris‐HCI (pH 7.5), 10 mM magnesium acetate, 0.2 mg·ml‐1 histone, and 1 μM Ca2+. The amounts of phospholipid (phosphatidylserine (PS)/ dipalmitoylphosphatidylcholine (DPPC), 1:4 molar ratio), TPA, and local anesthetics used were 100 μM, 100 nM and 0.5 mM, respectively. These results indicated that lidocaine and other local anesthetics inhibited PKC activity, with dibucaine shown to be the most potent inhibitor.

#### **4. Novel method for determining antioxidant activity of medical agents**

We developed a phycoerythrin fluorescence‐based assay to determine the antioxidant activ‐ ity of various medical agents including local anesthetics [3, 16, 17]. This assay system consists of B‐phycoerythrin (B‐PE) as a fluorescence molecule to show oxidative stress and 2,2′‐azo‐ bis (2‐amidinopropane) dihydrochloride (AAPH) as a hydrophilic oxidative stress simulator, which continuously generates peroxyl radicals at a constant rate, making it possible to easily evaluate the antioxidant activities of various medical agents [18]. Since the system is based on protein oxidation by peroxyl radicals, it is considered to be a model of in vivo ROS reactions. The detailed reactions of B‐PE with AAPH can be illustrated as follows.

$$\text{R}-\text{N}=\text{N}-\text{R}\rightarrow\text{R}\bullet+\text{N}\_{2}+\bullet\\\text{R}\rightarrow(\text{1}-\text{e})\text{ R}-\text{R}\tag{1}$$

$$
\bigvee \text{ 2eR} \bullet \tag{2}
$$

$$\mathsf{R}\bullet\mathsf{+O}\_{\mathsf{z}}\rightarrow\mathsf{RO}\_{\mathsf{z}}\bullet\tag{3}$$

$$\text{RO}\_2 \bullet \text{-}\mathsf{B}-\mathsf{PE} \to \text{ stable products} \tag{4}$$

Regional Anesthesia: Advantages of Combined Use with General Anesthesia and Useful Tips for Improving... http://dx.doi.org/10.5772/66573 33

$$\text{RO}\_2\bullet \text{ + A \to stable products}\tag{5}$$

The structure of AAPH [HCl•NH=C(NH<sup>2</sup> )‐C(CH3 )2 ‐N=N‐C(CH<sup>3</sup> )2 ‐C(NH<sup>2</sup> )=NH•HCl] is represented by "R‐N=N‐R", where "e" represents the efficiency of free radical generation and "A" is an antioxidant. AAPH undergoes thermal decomposition to yield free radi‐ cals (reaction (2)), which rapidly react with oxygen molecules nearby to produce peroxyl radicals (reaction (3)). With this assay, peroxyl radicals may attack B‐PE, resulting in fluo‐ rescence decay (reaction (4)), which, if successful, may be scavenged by an antioxidant (reaction (5)).

**4. Novel method for determining antioxidant activity of medical agents**

The detailed reactions of B‐PE with AAPH can be illustrated as follows.

R • +O2 → RO2

10 mM magnesium acetate, 0.2 mg·ml‐1

32 Current Topics in Anesthesiology

dibucaine shown to be the most potent inhibitor.

We developed a phycoerythrin fluorescence‐based assay to determine the antioxidant activ‐ ity of various medical agents including local anesthetics [3, 16, 17]. This assay system consists of B‐phycoerythrin (B‐PE) as a fluorescence molecule to show oxidative stress and 2,2′‐azo‐ bis (2‐amidinopropane) dihydrochloride (AAPH) as a hydrophilic oxidative stress simulator, which continuously generates peroxyl radicals at a constant rate, making it possible to easily evaluate the antioxidant activities of various medical agents [18]. Since the system is based on protein oxidation by peroxyl radicals, it is considered to be a model of in vivo ROS reactions.

**Figure 8.** Effects of lidocaine and other local anesthetics on PKC activity in rat brain. PKC was obtained from a rat brain and purified using a method previously described [10]. Enzyme activity was assessed by measuring the incorporation of 32P from [γ‐<sup>32</sup>P]ATP into H1 histone (type IIIs) at 30°C for 3 min. The medium contained 20 mM Tris‐HCI (pH 7.5),

dipalmitoylphosphatidylcholine (DPPC), 1:4 molar ratio), TPA, and local anesthetics used were 100 μM, 100 nM and 0.5 mM, respectively. These results indicated that lidocaine and other local anesthetics inhibited PKC activity, with

histone, and 1 μM Ca2+. The amounts of phospholipid (phosphatidylserine (PS)/

 R − N = N − R → R • +N<sup>2</sup> + •R → (1 − e) R − R (1) ↘ 2eR• (2)

RO2 • +B − PE → stable products (4)

• (3)

**Figure 9.** *Left panel*: Typical decay of fluorescence of B‐PE (B‐phycoerythrin) with AAPH (2,2′‐azobis (2‐amidinopropane) dihydrochloride) in Tris‐HCI buffer (pH 7.4) at 38°C in the absence and presence of Trolox. The amounts of B‐PE, AAPH, Trolox, and Tris‐HCl buffer used were 1.78 nM, 6.25 mM, 4 μM, and 40 mM, respectively. The fluorescence excitation and emission wavelength were 545 nm (3‐nm slit) and 575 nm (5‐nm slit), respectively. Exposure of B‐PE to peroxyl radicals generated by AAPH led to a decrease in B‐PE fluorescence. This peroxidation process was efficiently inhibited by addition of Trolox, which has potent antioxidant activity. The characteristic features of this assay offer great advantages for determining the possible antioxidant activity of various compounds when added to the reaction mixture. *Right panel*: Effects of lidocaine on B‐PE fluorescence decay. Although lidocaine did not completely abrogate the fluorescence decay of B‐PE, it reduced the rate of decay in a dose‐dependent manner. This finding indicated that lidocaine has an antioxidant function, though its potency is not as strong as that of Trolox.

The rate of peroxyl radical generation, R, from AAPH at a constant temperature is shown by Eq. (6) [19]:

$$\mathbf{R} = \mathbf{K} \times \text{[AAPH]} \tag{6}$$

where K is the rate constant for radical generation from AAPH and [AAPH] is the concentra‐ tion of AAPH in M. The rate of radical generation is virtually constant during the first few hours of this assay [20], since the half‐life of AAPH is approximately 175 h in neutral pH water at 37°C [19]. The rate of peroxyl radical generation at 38°C under the present assay conditions was 1.56 × 10‐6  × [AAPH] (M•s‐1 ) [3].

B‐PE is a multisubunit protein extracted from the unicellular red alga, *Porphyridium cruentum* [16, 20]. Since it is easily oxidized, which decreases its fluorescence, B‐PE functions as a reporting molecule of oxidative stress induced by peroxyl radicals from AAPH. In addition, because of its very high extinction coefficient and fluorescence quantum yields, B‐PE can be readily detected by fluorescence spectroscopy at concentrations as low as 10‐12 M [20].

For this assay, the fluorescence decay of B‐PE by the AAPH‐generated peroxyl radical was spectrophotometrically monitored at an excitation of 545 nm (3‐nm slit) and emission of 575 nm (5‐nm slit). The reaction mixture (2 ml) contained 1.78 nM B‐PE and 6.25 mM AAPH in 40 mM Tris‐HCl buffer (pH 7.4) at 38°C. Since the system is not closed, oxygen for the reac‐ tions is freely supplied from the atmosphere through the surface of the reaction mixture. As shown in **Figure 9**, B‐PE fluorescence was linearly decreased by exposure to AAPH, which has a linear relationship with B‐PE concentration. This peroxidative destruction of B‐PE can be temporarily stopped by addition of a typical radical scavenger, such as Trolox.
