Iw .I Radmon Oncology Rio/ Phvv. Vol. 20. pp. 59-64 Printed ,n the U.S.A. All rights reserved.
??Original Contribution
EFFECT
OF EXTERNAL pH ON HEAT SENSITIZATION BY LOCAL ANESTHETICS
RONALD A. Coss, Laboratory
PH.D.
AND NANCY N. SMITH, M.S.
of Experimental Radiation Oncology, Department of Radiation Oncology and Nuclear Medicine, Thomas Jefferson University Hospital, 1020 Sansom St., Philadelphia, PA 19107-5004
The sensitization of asynchronous Chinese hamster ovary (CHO) cells to 43°C by procaine, lidocaine, and tetracaine was examined, with the pH of the medium carefully controlled at xpH7 and 8. Thermal enhancement factors for 43°C were calculated for the surviving fraction of 0.01. The thermal enhancement factors of the local anesthetics were increased at =pHS by factors of 2-12, depending on the local anesthetic and its concentration. The concentration of the uncharged free base form of the local anesthetic in the culture medium correlated positively with the thermal enhancement factors of each local anesthetic and in a near linear fashion with the thermal enhancement factors for procaine. However, the concentration of the cationic form of the local anesthetics in the growth medium did not correlate with heat sensitization. We conclude that the ability of local anesthetics to sensitize cells to heat killing is dramatically influenced by the extracellular pH, with increased sensitization at the more basic pH’s. Secondly,
it is the extraceiiuiar concentration of the free -baseform of the iocai anesthetics that correiates with heat sensitization. Hyperthermia,
Local anesthetics,
pH dependence, Cytotoxicity.
taken from tables calculated for 22.5-30°C. When the pK,s for the LA at 43°C were used in the [R] and [RH+] calculations, only the [R] of the LA in the external medium was found to correlate with the thermal enhancement factor (TEF) in a positive manner.
INTRODUCIION Local anesthetics (LA) sensitize both prokaryotes and eukaryotes to heat killing (5,6, 18,20). The objective of this
study was to determine if a relationship exists between the efficacy of LA to sensitize mammalian cells to heat killing and the concentration of the charged, cationic form (RH+) or the uncharged, free base form (R) of the LA. A dose response relationship between heat sensitization and the [RH+] or the [R] may indicate the mechanism of sensitization by LA. The RHf form displaces Ca2+ from bound membrane sites (2, 15, 16). The R form fluidizes membranes and induces release of Ca2+ from membrane sequestered sites ( 1, 14, 16).
METHODS
AND
MATERIALS
CHO 10B4 cells were maintained in exponential growth at 37’C, 5% CO2 in Falcon plastic tissue culture flasks by subculturing 3 times weekly in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS). Cells were inoculated into spinner flasks containing McCoy’s 5A with tnoz.
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of Chinese hamster ovary (CHO) cells by LA, a consistent relationship was not found between sensitization and the [R] or [RH+] of the LA (4). The pH was not carefully controlled in this previous study. When we repeated these experiments and maintained the extracellular pH at -7 and =8, we observed a dramatic and reproducible increase in cytotoxicity at the higher pH. Furthermore, in the previous study, the pK,s used in the calculations were
On the day of an experiment, cells were concentrated and resuspended in McCoy’s 5A without FBS. Furthermore, this resuspension medium [5A(H + M)] was buffered with 10 mM 4-(2_hydroxyethyl)- I -piperazineethanesulfonic acid (HEPES) and 10 mM 2-(N-morpho1ino)ethanesulfonic acid (MES) (11) to the pH appropriate for the experiment. Sodium bicarbonate was omitted from the 5A(H + M). Concentrated solutions of procaine HCl*,
This work was presented in part at Symposium on Hyperthermic Oncology, gust-3 September 1988. Reprint requests to: Ronald A. Goss, Acknowledgments-The authors thank Xi-Lian Li for suggesting that we control and John J. Hayes, Jr., for bringing the
attention. The authors thank Taylor and Francis, Inc., for permission to include variations of two figures they had previously published. This work was supported by grant No. CA 38656 awarded by the National Cancer Institute, National Institutes of Health, Department of Health and Human Services to R.A.C. Accepted for publication 26 April 1990. * Sigma, St. Louis, MO.
the Fifth International Kyoto, Japan, 29 AuPh.D. William C. Dewey and the pH of the medium, pK, calculations to our
59
60
I. J. Radiation Oncology 0 Biology 0 Physics
lidocaine HCl,+ and tetracaine HCl* were prepared in 5A(H + M) at the appropriate pH. The solutions were filter sterilized prior to addition to the spinner flasks. Exposure to heat (43°C) + LA was performed in spinner flasks containing 50 ml 5A(H + M). Spinners were transfered to a 43°C water bath to allow for temperature equilibration prior to addition of cells. Drug solutions were added 15 min prior to inoculation of cells. Control samples ^_.^^_CrL..c --..:111--^L:-- I_ A___.WG1G . .. . . ,,,,,n,,.~ ,.I-_..^ CXccyr LU urug, IJ’“c;eJscu ^_ i13auuvc, Lila1cqulllu1auu11 etc. was performed in a 37°C incubator. The CHO cells were inoculated into preheated and control spinner flasks at a final concentration of 200,000 cells/ml, and exposure times were determined from time of inoculation. Aliquots of cells were removed after varying times of heating and plated into 60 mm petri dishes containing irradiated feeder cells ( 10) in McCoys 5A Medium + 10% FBS. The dishes were stained 7-10 days later and colonies counted. The TEF (TEF = Do, 43”C/Do, 43°C + LA) was calculated using DO,Ssince they incorporated reductions of both D, and D,. The following variation of the Henderson-Hasselbalch equation was used to calculate the [R] and [RH+] of the LAS:
hd[Rl/[RH+l) = PH
- pk.
The medium was measured with a temperature _~~_DH ran of _~the ~___ ~~~_ __..~~~ controlled pH meterS equipped with a microprobe electrode.” The pH of the medium in the spinners was measured before the addition of cells and at the termination of the experiment. The pH indicated in parentheses on the survival curves is the pH at the termination of heating and is that used in the calculations of [R] and [RH+]. RESULTS The influence of pH7 and pH8 on the molar efficacy ,.F.....,.,.“:..n 1:rl,.,,,:,, ,...A +,c..,.,-.,.:.., I.,,* “,w.,.:c:,,L” p’vuuus;, IIU”C(IIIIG, QUU LGL1aLallK) as llCdL JCLl~~llldC;‘~
“1
was examined. These pH’s were selected because they would significantly alter the concentrations of the acid and base forms of the LA. Secondly, short exposures at 37” to these pH values did not affect the plating efficiency of the cells. The p&s for procaine, lidocaine, and tetraCaine at 43°C were determined to be 8.69,7.60 and 8.25, respectively ( 12, 13). These pK,s were used in all subsequent calculations. Two typical experiments illustrating the influence of external pH on the molar efficacy of procaine HCl as a sensitizer of 43°C hyperthermia are shown in Figures 1A and 1B. The pH of the spinner flasks in Figure 1A varied between 6.74 and 6.85, whereas the pH of the spinner flasks in Figure 1B varied between 7.71 and 7.78. Both
+ ~nvenex, r_..___.. umw T-IL__
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Spring-
January 199 I, Volume 20, Number I
the shoulder and the slope of the survival curves decreased with increasing drug concentration for both pH ranges. However, the heat sensitization was greater for the more basic pH range. The enhanced sensitization by procaine HCl at the basic pH range is illustrated convincingly in Figure 1C. In this figure the TEF at the surviving fraction (SF) of 0.0 1 is plotted against the drug concentration for the two pH ranges. ‘p1-_,____-L_~L:_-_ _Cr,-_ __:_I __J L,__ I?____ _c.-__ 1ne LuIlcaltrdlluns 01 lne dLXU drlU DdSt:IUrmS 01 pruCaine HCl were calculated and compared to the TEF for all pHs tested using the Henderson-Hasselbalch equation given in the Methods and Materials section. The result of these calculations are graphically shown in Figures 1D and 1E. A positive linear relationship exists between the [R] of procaine HCl in the external medium and heat sensitization (Figure 1D). However, the [RH+] of procaine HCl in the external medium does not correlate with TEF (Figure 1E). The above experiments were repeated for lidocaine and tetracaine. The TEF is plotted against drug concentration for the acid and basic pH ranges in Figure 2A (lidocaine) and Figure 3A (tetracaine). Significantly enhanced heat sensitization was observed in the basic pH ranges for both LA. Tetracaine was the most potent heat sensitizer. As was observed for procaine, when the [R] and [RH+] forms of these drugs __-_ were ..--- nlotted =____-_ against ----, the -x2------ the ---- TEF. I onlv ---- IRl L--J in the external medium correlated positively with TEF (see Figure 2B for lidocaine and Figure 3B for tetracaine). We were interested in whether consistent correlations existed between the concentration of the acid or free base form of the LA inside of the cells, [RH+]i and [R]i, respectively, and the TEF. The concentration of the R form inside of the cell was assumed to be the concentration of the R form in the external medium. The internal pH at 37°C in the presence of HEPES and MES in the external medium was calculated as a function of the external pH ,.-.I r\_._.^_. I?\ c-_ PUA ?.-I,- .I,,. I?_-A-4.. “I _l?PL.. ll”lll dl-LllCllilta L‘lll ilull ucwcy (3, I”‘ LIl” c;CUS.WC assumed that the LA would not change the internal pH (7 mM procaine does not change the internal pH in CHO cells {W. C. Dewey, oral communication, January, 19893). These values were used in the modified Henderson-Hasselbalch equation to estimate the minimal concentration of the RH+ form of the anesthetic inside of the cell. The calculated [RH+]i was then plotted against the TEF. The results are presented in Figures 4A-C for procaine, lidocaine, and tetracaine. The estimated [RH+]i values generally exhibited positive correlations with increasing TEF for all three LA. One of the most dramatic results of these calculations is that for procaine and tetracaine the concentration of the RH+ form inside the cell accumulates to a much higher concentration than that in
4 Extra-iong Caiomei Fisher Scientific.
MicroProbe
Combination
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pH dependency ofheatsensitizationby localanesthetics0R.A. COSS AND N. N. SMITH
61
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Fig. I. The effect of procaine (P) treatment on hyperthermic killing of CHO cells at 43°C and its modification by the pH of the external medium. A: example of a single experiment demonstrating the concentration dependent potentiation of 43°C killing by procaine at slightly acid pH (pH values are given in parentheses). The sampling error (standard deviation) in this figure and Figure 1B are shown only when larger than the symbols. (Variation of Figure 1 from reference (7) B: example of a single experiment demonstrating the influence of basic pH on the concentration dependent potentiation of 43’C killing by procaine. (Variation of Figure 2 from reference (7). C: pH dependent sensitization by procaine expressed as TEF at the 0.01 SF vs. the concentration of the LA. Note the dramatically enhanced sensitization for the pH 7.75-8.00 range compared to the pH 6.7 l-7.00 range. Error bars (standard deviations of the mean) are given only when larger than the symbols in this figure and Figures 2A and 3A. D: comparison of the calculated concentration of the free base form of the LA in the external medium, [R]e, with the TEF. The data represent individual points from four experiments and for both the acid and basic pHs. Note the linear relationship. E: comparison of the calculated concentration of the charged, cationic form of the LA in the external medium, [RH+],, with the corresponding TEF. The data are derived from the same experiments used in Figure 1D. Note that the data are scattered and do not exhibit any relationship. (Figures 1A and 1B are variations of Figures I and 2 from reference (7).
1. J. Radiation Oncology 0 Biology 0 Physics
62
January 1991, Volume 20, Number I 5
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[R]e mM
Fig. 2. The effect of lidocaine (L) treatment on hyperthermic killing of CHO cells at 43°C and its modification by the pH of the external medium. A and B: as for Figures lC-D, except for lidocaine.
the extracellular
medium. The calculated concentrations are so high for the charged form of procaine that one would expect the internal pH to be affected. As mentioned
previously, we had assumed that the LA would not affect the internal pH. However, these calculations indicate that the internal pH definitely needs to be measured. Similar calculations were performed using internal pH values based on the data of Fellenz and Gerweck (9) for CHO cells. Again, although the [RH+]i values were slightly different, similar correlations were found (data not shown). DISCUSSION Yau and Kim (2 1) and Yatvin et al. (19) have reported on the variable potency of LA as heat sensitizers. Yau and Kim used murine L5 178Y cells to examine the molar effectiveness of dibucaine, lidocaine, tetracaine, marcaine, and procaine. They found that dibucaine was the most effective and procaine the least effective of the group. Yatvin et al. used a bacterial strain (E. coli K 1060) to compare heat sensitization by procaine, lidocaine, tetracaine, and benzocaine with the anesthetic potency of these drugs. They found that tetracaine was the most effective and
procaine the least effective, on a molar basis, as heat sensitizers. Furthermore, the Yatvin group found a linear correlation between the anesthetic potency of the drugs (for inducing respiratory arrest and modifying myocardial contractile force) and their ability to sensitize bacteria to heat killing. We asked if any consistent relationship exists between the molar concentration of the LA, especially of their charged and uncharged forms, and their effectiveness as heat sensitizers of mammalian cells. We observed that the concentration of the uncharged, free base form of the LA in the external medium, [RI,, correlated in a positive manner with heat sensitization, whereas the concentration of the cationic form of the LA, [RH+],, did not. This implies that the binding of the cationic form to external membrane proteins and/or displacement of bound Ca*+ from the outside of the plasma membrane (2) does not play a significant role in heat sensitization. The uncharged free base form is lipid soluble, fluidizes the plasma membrane, and rapidly enters cells via the plasma membrane. The R form subsequently establishes an equilibrium with the RH+ form inside the cell ( 17). The importance of the [Rle agrees with the findings of Yatvin et al. ( 19) correlating the “equivalent killing
B Tetracaine ??
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Fig. 3. The effect of tetracaine (T) treatment on the hyperthermic killing of CHO cells at 43°C and its modification by the pH of the external medium. A and B: as for Figures IC-D, except for tetracaine.
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pH dependency of heat sensitization by local anesthetics 0 R. A.
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Fig. 4. Comparison of the calculated intracellular concentration of the cationic form, [RH+]i, of the LA with its corresponding TEF. A, procaine; B, lidocaine; and C, tetracaine. Note the positive relationships for the [RH+]i and TEF of procaine and tetracaine, and that the calculated [RH+]i values can be much larger than the extracellular concentrations. Also note the inconsistency in some of the calculated [RH+]i values for lidocaine and the corresponding TEFs, especially in the 3.5-4.5 mM range.
COSS AND
N. N. SMITH
63
dose” (bacteria) to the anesthetic potency in mammals. The concentration of the extracellular free base form would dictate the physiological responses through a cascade effect. However, the importance of [Rle does not indicate whether or not sensitization of mammalian cells to heat follows predominantly from: (a) alterations in plasma membrane fluidity by R; (b) modification of the integrity of cytoplasmic membranes by intracellular R; or (c) disruption of Ca2+-homeostasis by intracellular RH+. Can a statement be made about the molar effectiveness of LA and the concentration of the acid and base forms inside of mammalian cells? We calculated what these values might be for CHO cells using the concentration of the free base form in the extracellular medium as the starting point for determining [RH+]i. Positive correlations were found when either [Rle ([Rle = [R]i) or [RH+]i were plotted against TEF for procaine and tetracaine. The relationship was not always consistent for the [RH+]i of lidocaine. The TER for comparable [RH+]i values of lidocaine were different depending on whether the cells were heated at the acid or basic pH ranges (see Figure 4B, [RH+]i values between 3.5-4.5 mM). These discrepancies probably follow from using incorrect intracellular pH values at the treatment temperature. We are aware that these are calculated values, and are minimal values that do not take into account sequestration of the RH+ form. Actual measurements need to be performed of the internal pH in the presence of the LA at 43°C. ** However, the data suggest that both intracellular forms of the LA may play roles in modifying the heat sensitivity of the CHO cells. In other words, modification of membrane fluidity and intracellular free calcium levels may each play a role in heat sensitization by the LA. In addition, as Dewey (8) notes, heat sensitization may possibly result from the charged form of the anesthetic interacting directly with intracellular proteins, increasing their sensitivity to denaturation. In conclusion, we demonstrate that the extracellular concentration of the free base form of the LA correlates with heat sensitization. The external pH greatly modifies the heat sensitization by LA, and this is directly traceable to the concentration of the R form of the LA in the external medium. Therefore one must carefully control the pH when performing experiments using combinations of heat and LA in order to achieve reproducible results.
REFERENCES Bianchi, C. P. Cellular pharmacology of contraction of skeleta1 muscle. In: Narahashi, T., ed. Cellular pharmacology of excitable tissue. Springfield, OH: Thomas; 1975:485-5 19. 2. Campbell, A. K. Intracellular calcium. New York: John Wiley & Sons Ltd.; 1983:433-437. 1.
** Indeed, J. R. Dynlacht, Colorado State University, informed me orally (March, 1990) that 10 mM procaine reduced the internal pH of CHO cells when combined with 45°C.
3. Chu, G. L.; Dewey, W. C. The role of low intracellular or extracellular pH in sensitization to hyperthermia. Radiat. Res. 114:154-167; 1988. 4. Coss, R. A. Correlation of heat sensitization of CHO cells by local anesthetics with the concentration of their cationic
64
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6.
7.
8. 9.
10.
11.
12.
13.
I. J. Radiation Oncology ?? Biology 0 Physics
and free base forms (Abstr.). 34th Annual Meeting of the Radiat. Res. Society; 1986:73. Goss, R. A.; Dewey, W. C. Heat sensitization of Cl- and Sphase cells by procaine hydrochloride. Radiat. Res. 92:6 15617; 1982. Coss, R. A.; Dewey, W. C. Heat sensitization of Cl and S phase cells by procaine HCl. II. Toxicity and probability of dividing following treatment. Int. J. Hyper. 4:687-697; 1988. Coss, R. A.; Smith, N. N. Heat sensitization by local anesthetics is strongly influenced by pH. In: Sugahara, T., Saito, M., eds. Hyperthermic oncology 1988, Vol. 1. Summary papers. New York: Taylor and Francis; 1989:268-269. Dewey, W. C. The search for critical cellular targets damaged by heat. Radiat. Res. 120: 19 l-204; 1989. Fellenz, M. P.; Gerweck, L. E. Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity. Radiat. Res. 116:305-312; 1988. Highfield, D. P.; Holahan, E. V.; Holahan, P. K.; Dewey, W. C. Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating. Radiat. Res. 97: 139- 153; 1984. Hofer, K. G.; Mivechi, N. F. Tumor cell sensitivity to hyperthermia as a function of extracellular and intracellular pH. J. N. C. I. 65:621-625; 1980. Kamaya H.; Hayes, J. J., Jr.; Ueda, I. Dissociation constants of local anesthetics and their temperature dependence. Anesth. Analg. 62:1025-1030; 1983. Kamaya, H.; Hayes, J. J., Jr.; Ueda, I. Errata. Anesth. Analg. 63:703; 1984.
January 199I, Volume 20, Number I
14. Lakshminarayanaiah, H.; Bianchi, C. P. Local anaesthetics. In: Goldberg, P. B., Roberts, J., eds. Handbook on pharmacology of aging. Boca Raton, FL: CRC Press; 1983: 127153. 15. Low, P. S.; Lloyd, D. H.; Stein, T. M.; Rogers, J. A. Calcium displacement by local anesthetics. Dependence on pH and anesthetic charge. J. Biol. Chem. 254:4119-4125; 1979. 16. Papahadjopoulos, D. Studies on the mechanism of action of local anesthetics with phospholipid model membranes. B. B. A. 265:169-186; 1972. 17. Ritchie, J. M. An overview of the mechanisms of local anesthetic action, past, present and future. In: Roth, S. H., Miller, K. W., eds. Molecular and cellular mechanisms of anesthetics. New York: Plenum Medical Book Co.; 1983: 191-202. 18. Yatvin, M. B. The influence of membrane lipid composition and procaine on hyperthermic death of cells. Int. J. Radiat. Biol. 32:5 13-52 1; 1977. 19. Yatvin, M. B.; Gipp, J. J.; Rusy, B. F.; Dennis, W. H. Correlation of bacterial hyperthermic survival with anaesthetic potency. Int. J. Radiat. Biol. 42:141-149; 1982. 20. Yau, T. M. Procaine-mediated modification of membranes and the response to X-irradiation and hyperthermia in mammalian cells. Radiat. Res. 80:523-54 1; 1979. 21 Yau, T. M.; Kim, S. C. Local anaesthetics as hypoxic radiosensitizers, oxic radioprotectors and potentiators of hyperthermic killing in mammalian cells. Br. J. Radiol. 53: 687-692; 1980.
??Original Contribution
EFFECT
OF EXTERNAL pH ON HEAT SENSITIZATION BY LOCAL ANESTHETICS
RONALD A. Coss, Laboratory
PH.D.
AND NANCY N. SMITH, M.S.
of Experimental Radiation Oncology, Department of Radiation Oncology and Nuclear Medicine, Thomas Jefferson University Hospital, 1020 Sansom St., Philadelphia, PA 19107-5004
The sensitization of asynchronous Chinese hamster ovary (CHO) cells to 43°C by procaine, lidocaine, and tetracaine was examined, with the pH of the medium carefully controlled at xpH7 and 8. Thermal enhancement factors for 43°C were calculated for the surviving fraction of 0.01. The thermal enhancement factors of the local anesthetics were increased at =pHS by factors of 2-12, depending on the local anesthetic and its concentration. The concentration of the uncharged free base form of the local anesthetic in the culture medium correlated positively with the thermal enhancement factors of each local anesthetic and in a near linear fashion with the thermal enhancement factors for procaine. However, the concentration of the cationic form of the local anesthetics in the growth medium did not correlate with heat sensitization. We conclude that the ability of local anesthetics to sensitize cells to heat killing is dramatically influenced by the extracellular pH, with increased sensitization at the more basic pH’s. Secondly,
it is the extraceiiuiar concentration of the free -baseform of the iocai anesthetics that correiates with heat sensitization. Hyperthermia,
Local anesthetics,
pH dependence, Cytotoxicity.
taken from tables calculated for 22.5-30°C. When the pK,s for the LA at 43°C were used in the [R] and [RH+] calculations, only the [R] of the LA in the external medium was found to correlate with the thermal enhancement factor (TEF) in a positive manner.
INTRODUCIION Local anesthetics (LA) sensitize both prokaryotes and eukaryotes to heat killing (5,6, 18,20). The objective of this
study was to determine if a relationship exists between the efficacy of LA to sensitize mammalian cells to heat killing and the concentration of the charged, cationic form (RH+) or the uncharged, free base form (R) of the LA. A dose response relationship between heat sensitization and the [RH+] or the [R] may indicate the mechanism of sensitization by LA. The RHf form displaces Ca2+ from bound membrane sites (2, 15, 16). The R form fluidizes membranes and induces release of Ca2+ from membrane sequestered sites ( 1, 14, 16).
METHODS
AND
MATERIALS
CHO 10B4 cells were maintained in exponential growth at 37’C, 5% CO2 in Falcon plastic tissue culture flasks by subculturing 3 times weekly in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS). Cells were inoculated into spinner flasks containing McCoy’s 5A with tnoz.
l”l”
EDC I u&J
I ,... 1 “1
7 An.rr .L uay3
..An, p”“’
?? rr
nm .3”..,xr;-,m.* L” au ~*pGlullG,,r.
of Chinese hamster ovary (CHO) cells by LA, a consistent relationship was not found between sensitization and the [R] or [RH+] of the LA (4). The pH was not carefully controlled in this previous study. When we repeated these experiments and maintained the extracellular pH at -7 and =8, we observed a dramatic and reproducible increase in cytotoxicity at the higher pH. Furthermore, in the previous study, the pK,s used in the calculations were
On the day of an experiment, cells were concentrated and resuspended in McCoy’s 5A without FBS. Furthermore, this resuspension medium [5A(H + M)] was buffered with 10 mM 4-(2_hydroxyethyl)- I -piperazineethanesulfonic acid (HEPES) and 10 mM 2-(N-morpho1ino)ethanesulfonic acid (MES) (11) to the pH appropriate for the experiment. Sodium bicarbonate was omitted from the 5A(H + M). Concentrated solutions of procaine HCl*,
This work was presented in part at Symposium on Hyperthermic Oncology, gust-3 September 1988. Reprint requests to: Ronald A. Goss, Acknowledgments-The authors thank Xi-Lian Li for suggesting that we control and John J. Hayes, Jr., for bringing the
attention. The authors thank Taylor and Francis, Inc., for permission to include variations of two figures they had previously published. This work was supported by grant No. CA 38656 awarded by the National Cancer Institute, National Institutes of Health, Department of Health and Human Services to R.A.C. Accepted for publication 26 April 1990. * Sigma, St. Louis, MO.
the Fifth International Kyoto, Japan, 29 AuPh.D. William C. Dewey and the pH of the medium, pK, calculations to our
59
60
I. J. Radiation Oncology 0 Biology 0 Physics
lidocaine HCl,+ and tetracaine HCl* were prepared in 5A(H + M) at the appropriate pH. The solutions were filter sterilized prior to addition to the spinner flasks. Exposure to heat (43°C) + LA was performed in spinner flasks containing 50 ml 5A(H + M). Spinners were transfered to a 43°C water bath to allow for temperature equilibration prior to addition of cells. Drug solutions were added 15 min prior to inoculation of cells. Control samples ^_.^^_CrL..c --..:111--^L:-- I_ A___.WG1G . .. . . ,,,,,n,,.~ ,.I-_..^ CXccyr LU urug, IJ’“c;eJscu ^_ i13auuvc, Lila1cqulllu1auu11 etc. was performed in a 37°C incubator. The CHO cells were inoculated into preheated and control spinner flasks at a final concentration of 200,000 cells/ml, and exposure times were determined from time of inoculation. Aliquots of cells were removed after varying times of heating and plated into 60 mm petri dishes containing irradiated feeder cells ( 10) in McCoys 5A Medium + 10% FBS. The dishes were stained 7-10 days later and colonies counted. The TEF (TEF = Do, 43”C/Do, 43°C + LA) was calculated using DO,Ssince they incorporated reductions of both D, and D,. The following variation of the Henderson-Hasselbalch equation was used to calculate the [R] and [RH+] of the LAS:
hd[Rl/[RH+l) = PH
- pk.
The medium was measured with a temperature _~~_DH ran of _~the ~___ ~~~_ __..~~~ controlled pH meterS equipped with a microprobe electrode.” The pH of the medium in the spinners was measured before the addition of cells and at the termination of the experiment. The pH indicated in parentheses on the survival curves is the pH at the termination of heating and is that used in the calculations of [R] and [RH+]. RESULTS The influence of pH7 and pH8 on the molar efficacy ,.F.....,.,.“:..n 1:rl,.,,,:,, ,...A +,c..,.,-.,.:.., I.,,* “,w.,.:c:,,L” p’vuuus;, IIU”C(IIIIG, QUU LGL1aLallK) as llCdL JCLl~~llldC;‘~
“1
was examined. These pH’s were selected because they would significantly alter the concentrations of the acid and base forms of the LA. Secondly, short exposures at 37” to these pH values did not affect the plating efficiency of the cells. The p&s for procaine, lidocaine, and tetraCaine at 43°C were determined to be 8.69,7.60 and 8.25, respectively ( 12, 13). These pK,s were used in all subsequent calculations. Two typical experiments illustrating the influence of external pH on the molar efficacy of procaine HCl as a sensitizer of 43°C hyperthermia are shown in Figures 1A and 1B. The pH of the spinner flasks in Figure 1A varied between 6.74 and 6.85, whereas the pH of the spinner flasks in Figure 1B varied between 7.71 and 7.78. Both
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January 199 I, Volume 20, Number I
the shoulder and the slope of the survival curves decreased with increasing drug concentration for both pH ranges. However, the heat sensitization was greater for the more basic pH range. The enhanced sensitization by procaine HCl at the basic pH range is illustrated convincingly in Figure 1C. In this figure the TEF at the surviving fraction (SF) of 0.0 1 is plotted against the drug concentration for the two pH ranges. ‘p1-_,____-L_~L:_-_ _Cr,-_ __:_I __J L,__ I?____ _c.-__ 1ne LuIlcaltrdlluns 01 lne dLXU drlU DdSt:IUrmS 01 pruCaine HCl were calculated and compared to the TEF for all pHs tested using the Henderson-Hasselbalch equation given in the Methods and Materials section. The result of these calculations are graphically shown in Figures 1D and 1E. A positive linear relationship exists between the [R] of procaine HCl in the external medium and heat sensitization (Figure 1D). However, the [RH+] of procaine HCl in the external medium does not correlate with TEF (Figure 1E). The above experiments were repeated for lidocaine and tetracaine. The TEF is plotted against drug concentration for the acid and basic pH ranges in Figure 2A (lidocaine) and Figure 3A (tetracaine). Significantly enhanced heat sensitization was observed in the basic pH ranges for both LA. Tetracaine was the most potent heat sensitizer. As was observed for procaine, when the [R] and [RH+] forms of these drugs __-_ were ..--- nlotted =____-_ against ----, the -x2------ the ---- TEF. I onlv ---- IRl L--J in the external medium correlated positively with TEF (see Figure 2B for lidocaine and Figure 3B for tetracaine). We were interested in whether consistent correlations existed between the concentration of the acid or free base form of the LA inside of the cells, [RH+]i and [R]i, respectively, and the TEF. The concentration of the R form inside of the cell was assumed to be the concentration of the R form in the external medium. The internal pH at 37°C in the presence of HEPES and MES in the external medium was calculated as a function of the external pH ,.-.I r\_._.^_. I?\ c-_ PUA ?.-I,- .I,,. I?_-A-4.. “I _l?PL.. ll”lll dl-LllCllilta L‘lll ilull ucwcy (3, I”‘ LIl” c;CUS.WC assumed that the LA would not change the internal pH (7 mM procaine does not change the internal pH in CHO cells {W. C. Dewey, oral communication, January, 19893). These values were used in the modified Henderson-Hasselbalch equation to estimate the minimal concentration of the RH+ form of the anesthetic inside of the cell. The calculated [RH+]i was then plotted against the TEF. The results are presented in Figures 4A-C for procaine, lidocaine, and tetracaine. The estimated [RH+]i values generally exhibited positive correlations with increasing TEF for all three LA. One of the most dramatic results of these calculations is that for procaine and tetracaine the concentration of the RH+ form inside the cell accumulates to a much higher concentration than that in
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Fig. I. The effect of procaine (P) treatment on hyperthermic killing of CHO cells at 43°C and its modification by the pH of the external medium. A: example of a single experiment demonstrating the concentration dependent potentiation of 43°C killing by procaine at slightly acid pH (pH values are given in parentheses). The sampling error (standard deviation) in this figure and Figure 1B are shown only when larger than the symbols. (Variation of Figure 1 from reference (7) B: example of a single experiment demonstrating the influence of basic pH on the concentration dependent potentiation of 43’C killing by procaine. (Variation of Figure 2 from reference (7). C: pH dependent sensitization by procaine expressed as TEF at the 0.01 SF vs. the concentration of the LA. Note the dramatically enhanced sensitization for the pH 7.75-8.00 range compared to the pH 6.7 l-7.00 range. Error bars (standard deviations of the mean) are given only when larger than the symbols in this figure and Figures 2A and 3A. D: comparison of the calculated concentration of the free base form of the LA in the external medium, [R]e, with the TEF. The data represent individual points from four experiments and for both the acid and basic pHs. Note the linear relationship. E: comparison of the calculated concentration of the charged, cationic form of the LA in the external medium, [RH+],, with the corresponding TEF. The data are derived from the same experiments used in Figure 1D. Note that the data are scattered and do not exhibit any relationship. (Figures 1A and 1B are variations of Figures I and 2 from reference (7).
1. J. Radiation Oncology 0 Biology 0 Physics
62
January 1991, Volume 20, Number I 5
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the extracellular
medium. The calculated concentrations are so high for the charged form of procaine that one would expect the internal pH to be affected. As mentioned
previously, we had assumed that the LA would not affect the internal pH. However, these calculations indicate that the internal pH definitely needs to be measured. Similar calculations were performed using internal pH values based on the data of Fellenz and Gerweck (9) for CHO cells. Again, although the [RH+]i values were slightly different, similar correlations were found (data not shown). DISCUSSION Yau and Kim (2 1) and Yatvin et al. (19) have reported on the variable potency of LA as heat sensitizers. Yau and Kim used murine L5 178Y cells to examine the molar effectiveness of dibucaine, lidocaine, tetracaine, marcaine, and procaine. They found that dibucaine was the most effective and procaine the least effective of the group. Yatvin et al. used a bacterial strain (E. coli K 1060) to compare heat sensitization by procaine, lidocaine, tetracaine, and benzocaine with the anesthetic potency of these drugs. They found that tetracaine was the most effective and
procaine the least effective, on a molar basis, as heat sensitizers. Furthermore, the Yatvin group found a linear correlation between the anesthetic potency of the drugs (for inducing respiratory arrest and modifying myocardial contractile force) and their ability to sensitize bacteria to heat killing. We asked if any consistent relationship exists between the molar concentration of the LA, especially of their charged and uncharged forms, and their effectiveness as heat sensitizers of mammalian cells. We observed that the concentration of the uncharged, free base form of the LA in the external medium, [RI,, correlated in a positive manner with heat sensitization, whereas the concentration of the cationic form of the LA, [RH+],, did not. This implies that the binding of the cationic form to external membrane proteins and/or displacement of bound Ca*+ from the outside of the plasma membrane (2) does not play a significant role in heat sensitization. The uncharged free base form is lipid soluble, fluidizes the plasma membrane, and rapidly enters cells via the plasma membrane. The R form subsequently establishes an equilibrium with the RH+ form inside the cell ( 17). The importance of the [Rle agrees with the findings of Yatvin et al. ( 19) correlating the “equivalent killing
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Fig. 4. Comparison of the calculated intracellular concentration of the cationic form, [RH+]i, of the LA with its corresponding TEF. A, procaine; B, lidocaine; and C, tetracaine. Note the positive relationships for the [RH+]i and TEF of procaine and tetracaine, and that the calculated [RH+]i values can be much larger than the extracellular concentrations. Also note the inconsistency in some of the calculated [RH+]i values for lidocaine and the corresponding TEFs, especially in the 3.5-4.5 mM range.
COSS AND
N. N. SMITH
63
dose” (bacteria) to the anesthetic potency in mammals. The concentration of the extracellular free base form would dictate the physiological responses through a cascade effect. However, the importance of [Rle does not indicate whether or not sensitization of mammalian cells to heat follows predominantly from: (a) alterations in plasma membrane fluidity by R; (b) modification of the integrity of cytoplasmic membranes by intracellular R; or (c) disruption of Ca2+-homeostasis by intracellular RH+. Can a statement be made about the molar effectiveness of LA and the concentration of the acid and base forms inside of mammalian cells? We calculated what these values might be for CHO cells using the concentration of the free base form in the extracellular medium as the starting point for determining [RH+]i. Positive correlations were found when either [Rle ([Rle = [R]i) or [RH+]i were plotted against TEF for procaine and tetracaine. The relationship was not always consistent for the [RH+]i of lidocaine. The TER for comparable [RH+]i values of lidocaine were different depending on whether the cells were heated at the acid or basic pH ranges (see Figure 4B, [RH+]i values between 3.5-4.5 mM). These discrepancies probably follow from using incorrect intracellular pH values at the treatment temperature. We are aware that these are calculated values, and are minimal values that do not take into account sequestration of the RH+ form. Actual measurements need to be performed of the internal pH in the presence of the LA at 43°C. ** However, the data suggest that both intracellular forms of the LA may play roles in modifying the heat sensitivity of the CHO cells. In other words, modification of membrane fluidity and intracellular free calcium levels may each play a role in heat sensitization by the LA. In addition, as Dewey (8) notes, heat sensitization may possibly result from the charged form of the anesthetic interacting directly with intracellular proteins, increasing their sensitivity to denaturation. In conclusion, we demonstrate that the extracellular concentration of the free base form of the LA correlates with heat sensitization. The external pH greatly modifies the heat sensitization by LA, and this is directly traceable to the concentration of the R form of the LA in the external medium. Therefore one must carefully control the pH when performing experiments using combinations of heat and LA in order to achieve reproducible results.
REFERENCES Bianchi, C. P. Cellular pharmacology of contraction of skeleta1 muscle. In: Narahashi, T., ed. Cellular pharmacology of excitable tissue. Springfield, OH: Thomas; 1975:485-5 19. 2. Campbell, A. K. Intracellular calcium. New York: John Wiley & Sons Ltd.; 1983:433-437. 1.
** Indeed, J. R. Dynlacht, Colorado State University, informed me orally (March, 1990) that 10 mM procaine reduced the internal pH of CHO cells when combined with 45°C.
3. Chu, G. L.; Dewey, W. C. The role of low intracellular or extracellular pH in sensitization to hyperthermia. Radiat. Res. 114:154-167; 1988. 4. Coss, R. A. Correlation of heat sensitization of CHO cells by local anesthetics with the concentration of their cationic
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8. 9.
10.
11.
12.
13.
I. J. Radiation Oncology ?? Biology 0 Physics
and free base forms (Abstr.). 34th Annual Meeting of the Radiat. Res. Society; 1986:73. Goss, R. A.; Dewey, W. C. Heat sensitization of Cl- and Sphase cells by procaine hydrochloride. Radiat. Res. 92:6 15617; 1982. Coss, R. A.; Dewey, W. C. Heat sensitization of Cl and S phase cells by procaine HCl. II. Toxicity and probability of dividing following treatment. Int. J. Hyper. 4:687-697; 1988. Coss, R. A.; Smith, N. N. Heat sensitization by local anesthetics is strongly influenced by pH. In: Sugahara, T., Saito, M., eds. Hyperthermic oncology 1988, Vol. 1. Summary papers. New York: Taylor and Francis; 1989:268-269. Dewey, W. C. The search for critical cellular targets damaged by heat. Radiat. Res. 120: 19 l-204; 1989. Fellenz, M. P.; Gerweck, L. E. Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity. Radiat. Res. 116:305-312; 1988. Highfield, D. P.; Holahan, E. V.; Holahan, P. K.; Dewey, W. C. Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating. Radiat. Res. 97: 139- 153; 1984. Hofer, K. G.; Mivechi, N. F. Tumor cell sensitivity to hyperthermia as a function of extracellular and intracellular pH. J. N. C. I. 65:621-625; 1980. Kamaya H.; Hayes, J. J., Jr.; Ueda, I. Dissociation constants of local anesthetics and their temperature dependence. Anesth. Analg. 62:1025-1030; 1983. Kamaya, H.; Hayes, J. J., Jr.; Ueda, I. Errata. Anesth. Analg. 63:703; 1984.
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14. Lakshminarayanaiah, H.; Bianchi, C. P. Local anaesthetics. In: Goldberg, P. B., Roberts, J., eds. Handbook on pharmacology of aging. Boca Raton, FL: CRC Press; 1983: 127153. 15. Low, P. S.; Lloyd, D. H.; Stein, T. M.; Rogers, J. A. Calcium displacement by local anesthetics. Dependence on pH and anesthetic charge. J. Biol. Chem. 254:4119-4125; 1979. 16. Papahadjopoulos, D. Studies on the mechanism of action of local anesthetics with phospholipid model membranes. B. B. A. 265:169-186; 1972. 17. Ritchie, J. M. An overview of the mechanisms of local anesthetic action, past, present and future. In: Roth, S. H., Miller, K. W., eds. Molecular and cellular mechanisms of anesthetics. New York: Plenum Medical Book Co.; 1983: 191-202. 18. Yatvin, M. B. The influence of membrane lipid composition and procaine on hyperthermic death of cells. Int. J. Radiat. Biol. 32:5 13-52 1; 1977. 19. Yatvin, M. B.; Gipp, J. J.; Rusy, B. F.; Dennis, W. H. Correlation of bacterial hyperthermic survival with anaesthetic potency. Int. J. Radiat. Biol. 42:141-149; 1982. 20. Yau, T. M. Procaine-mediated modification of membranes and the response to X-irradiation and hyperthermia in mammalian cells. Radiat. Res. 80:523-54 1; 1979. 21 Yau, T. M.; Kim, S. C. Local anaesthetics as hypoxic radiosensitizers, oxic radioprotectors and potentiators of hyperthermic killing in mammalian cells. Br. J. Radiol. 53: 687-692; 1980.
