The relationships between IC50 values and various physicochemical parameters of local anesthetic (molecular weight [MW], - log dissociation constant [pKa], and logarithm of partition coefficient [log P]) were analyzed using multiple regression analysis (log IC50 = -1.294 + 0.001 x MW - 0.009 x pKa - 0.532 x log P; r2 = 0.892; P < 0.05). Furthermore, stepwise regression analysis demonstrated that log P contributes greatly to control the IC50 value as shown in Figure 4 (log IC50 = -1.252 - 0.514 x log P; r2 = 0.891; P < 0.001). A significant negative correlation was also obtained between IC50 values and relative conduction blocking potency of local anesthetics (log IC50 = -2.171 - 0.084 x potency; r2 = 0.873; P < 0.001).
By treating neutrophils with either TTX (1 nM-1 micro M) or amiloride hydrochloride (100 nM-100 micro M), the chemiluminescence development induced by PMA was not affected. In addition, no effect was observed on the chemiluminescence development in the presence of VTD, an activator of Na+ channels, in the concentration range of 10 nM to 1 micro M (Figure 5). The presence of VTD 1 micro M did not affect lidocaine-induced inhibition of chemiluminescence development of neutrophils; maximum photon count using lidocaine 5 mM with VTD (191,569 +/- 4039) (n = 3) was not different from that using lidocaine 5 mM without VTD (190,280 +/- 13,428) (n = 3).
The present study demonstrated that all eight local anesthetics suppressed O2- generation of human neutrophils in a concentration-dependent manner between 0.1 mM and 10 mM. The IC50 value obtained for each local anesthetic in the present results correlated well with the logarithmic value of the partition coefficient (log P) obtained by n-octanol/water for each drug. The suppressive effects of local anesthetics on O2- generation have a rank order of potency similar to that reported for neuronal conduction block; the higher the lipid solubility of a local anesthetic, the greater the inhibition of O2- generation with the drug, even if it is an optical isomer, amino-ester, or amino-amide compound. The voltage-dependent, Na+ channel-modifying reagents TTX and VTD and the voltage-independent, Na+ current-modifying reagent amiloride were unlikely to affect O2- generation of human neutrophils. VTD did not appear to affect lidocaine-induced inhibition of O2- generation. These results suggest that the mechanism(s) for the suppression of O2- generation of neutrophils is (are) mediated predominantly by modification of membrane function and is (are) independent of Na (+) influx.
Although generally classified as nonexcitable cells, human neutrophils possess a variety of ion channels: K+, H+, Cl-, and nonselective cation channels . Local anesthetics act not only on the Na+ channel, but also on the K+ and Ca2+ channels of membranes of excitable cells such as neuronal and myocardial cells . For example, using isolated myocardial cells, lidocaine  and tetracaine  inhibit the voltage-dependent Na+ current and reduce the amplitudes of K+ and Ca2+ currents. Although the role of the Na+ channel of neutrophils is still not clear, the generation of O2- has been reported to be associated with changes of Na+ currents in exchange for Ca2+ and/or H+. Therefore, local anesthetics should exert the inhibitory effects on neutrophils through the blockade of ion channels.
To elucidate some of the mechanisms by which local anesthetics inhibit neutrophils' O2- generation, we examined the influences of three Na+ current-modifying reagents such as TTX, VTD, and amiloride on O2- generation. TTX inhibits voltage-dependent Na+ channels by binding at the extracellular surface of nerve membrane, occluding the channels by a mechanism separate from that of local anesthetics . VTD is a classic activator of the voltage-gated Na+ channel and binds to and selectively stabilizes an open state conformation of the Na+ channel, leading to a persistent increase in Na+ permeability in excitable tissues . Amiloride inhibits Na+ current rapidly and reversibly in nonexcitable tissue. Amiloride-sensitive Na+ channels are voltage independent, and their gating properties are regulated by hormones such as vasopressin, intracellular Ca (2+), and G proteins . However, we could not detect any effect of those three Na+ current-modifying reagents in the present experiments. Amiloride hydrochloride at 1 micro M-1 mM has been reported not to affect O2- generation stimulated by PMA in human neutrophils, despite an inhibition of Na+ influx . The Na+ channel activator VTD exerts the depolarizing effect on excitable cells by increasing resting Na+ permeability. The effect of VTD is inhibited completely by TTX, and VTD's action on the resting potential is inhibited by local anesthetics . It is also reported that 10 mM of lidocaine almost completely reverses depolarization induced by 1 mM of VTD in all fibers in the frog sciatic nerve . In the present experiments, however, the presence of VTD did not affect lidocaine's effect on O (2)- generation of neutrophils. Because of their nature as nonexcitable cells, human neutrophils are unlikely to have TTX-sensitive Na+ channels. Indeed, TTX did not affect O2- generation of neutrophils. Therefore, we can exclude the possibility that voltage-dependent Na+ channels and amiloride-blockable Na+ currents are involved in the inhibitory effects of local anesthetics on O2- generation of neutrophils stimulated by PMA in any important way.
Lidocaine inhibits protein kinase C (PKC) activity , and dibucaine and tetracaine inhibit NADPH-oxidase activity ; those are essential components for the O2- generation of neutrophils . The above three local anesthetics also affect the mobilization of intracellular Ca2+[4,5]. In the present experiments, we used PMA as a stimulant of neutrophils. PMA is characterized as a compound that directly activates PKC and induces the respiratory burst , an event during phagocytosis characterized by increased oxygen consumption of neutrophils, independently of the intracellular Ca2+ movements . Thus, although it is uncertain whether local anesthetics interact with the PMA binding site of the PKC system or not, the suppression of O2- generation of neutrophils seems unlikely to be a result of the interaction between the local anesthetic and PMA. In the present experiments, staurosporine, which inhibits PKC even with a low concentration , caused inhibition of O2- generation of neutrophils (Figure 5). Ca2+ channel antagonists such as verapamil inhibit O2- generation of human neutrophils, but their inhibition of O2- generation seems to be via mechanisms other than the blocking of Ca2+ influx . Consequently, it is not likely that Ca2+ influx principally affects the present results. The IC50 values of the present study agree with the results obtained by others who showed that the IC50 value of the O2- generation of neutrophils stimulated by PMA was 5 mM for lidocaine , 0.2 mM for dibucaine , and 0.6 mM for tetracaine .
Using ropivacaine, which is prepared as an S-(-)-enantiomer and a pharmacological active isomer , we could not observe a complete inhibition of O2- generation even with the highest concentration of 20 mM (final concentration) in the present study. A similar dose-response pattern for ropivacaine was observed in the prolongation of the QRS interval obtained with lidocaine and bupivacaine . Ropivacaine seems to act as a partial antagonist on O2- generation in neutrophils. At present, we may not exclude the possibility that the unique suppression observed with ropivacaine (enantiomer) might be responsible for its stereoselectivity of S-(-)-enantiomer and/or for its absence of R-(+)-enantiomer, and thus a lack of allosteric effect. It is also interesting in regard to their inhibitory action on neutrophils' O2- generation that eight local anesthetics seem to be divided into two groups according to Hill's coefficients: a low Hill's coefficient of 0.94-1.43 group (procaine, ropivacaine, mepivacaine, prilocaine, and lidocaine), and a high Hill's coefficient of 2.51-3.57 group (bupivacaine, dibucaine, and tetracaine). Since Hill's coefficients of compounds seem to indicate an allosteric binding characteristic or a cooperative action of local anesthetics , the membrane action of local anesthetics may be characterized with Hill's coefficients. In addition to membrane effect, it is reported in a recent article by Cederholm et al.  that amino-amide local anesthetics affect intracellular response and extracellular response of human neutrophils differently; prilocaine, being the least potent anesthetic observed in the present experiment, produced the most marked increase in the intracellular response accompanied by a reduction in extracellular responses of stimulated neutrophils. Furthermore, cocaine and its derivatives inhibit up-regulation of the receptor for C3bi, degranulation, and generation of O2-, and the order of potency of these drugs seems to be tetracaine > bupivacaine > cocaine > lidocaine. They seem to affect neutrophil function at sites distal to activation of PKC. A recent study has also shown that cyclic-3 prime,5 prime-adenosine monophosphate (cAMP) production of human lymphocytes in both the basal and the epinephrine-stimulated condition was suppressed by mepivacaine, ropivacaine, and bupivacaine; the suppression by bupivacaine was much greater than that by mepivacaine . Therefore, we cannot exclude the possibility that local anesthetics could affect the O2- generation of neutrophils more strongly in the intracellular phase, which should be more predominant in invading microorganisms, than in the extracellular phase.
Because of the high concentrations of local anesthetics needed to produce an inhibitory effect on human neutrophils, the present results may not provide a definite clinical implication. For example, if lidocaine were given for peripheral nerve block, its plasma concentration would result in an average of approximately 7 micro g/mL , which is equivalent to 25.8 micro M; such a high concentration could be found at the injection site of lidocaine. Local anesthetics suppress not only O2- generation of neutrophils but also their phagocytosis , metabolism , and lysosomal enzyme release [3,7]. The IC50 value of lidocaine obtained in the present study is 6.02 mM, which corresponds to 0.16% solution (1% lidocaine solution is equal to 36.9 mM). Therefore, it is conceivable that a locally injected local anesthetic with a high lipid solubility could affect the host defense mechanisms in the tissue, at least at the site of its injection. In contaminated wounds, a topical application of local anesthetic cream has been suggested to damage host defenses and to invite the development of infection . Neutrophils from patients with coronary disease receiving lidocaine for a long duration have been reported to have a significantly impaired ability to release O2-, even with its low plasma concentration level . Cocaine, which also has been reported to suppress aggregation, degranulation, and O2- generation of human neutrophils , was reported to cause infective endocarditis in intravenous cocaine addicts . The inhibition of O2- generation by local anesthetics, especially those with high lipid solubility, may cause critically ill patients to have a potentially increased susceptibility to infection.
In summary, the present study shows that all local anesthetics suppress O2- generation of human neutrophils in a concentration-dependent manner, and the suppressive effects correlate well with log P obtained by the n-octanol/water system. However, Na+ current-modifying reagents, such as TTX, VTD, and amiloride, do not have any effect. The high concentration of local anesthetics needed for suppression of neutrophils suggests that the action of local anesthetics on neutrophils' O2- generation is the outcome of the modification of the cell membrane, PKC, NADPH-oxidase, cAMP production, and protein phosphorylation through their membrane-lipid solubability.
The authors thank Dr. Kenichi Kohno for his excellent technical assistance and Dr. Shuichiro Ohta, MD, for his constant support. The authors also thank Professor Toshihiko Uematsu, MD, PhD, for insightful discussion.
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