Download Lipid Solubility Modulates pH Potentiation of Local Anesthetic Block

513
Lipid Solubility Modulates pH Potentiation of Local
Anesthetic Block of Vmax Reactivation in Guinea Pig
Myocardium
Archer Broughton, Augustus O. Grant, C. Frank Starmer, Jayne K. Klinger, Bruce S. Stambler,
and Harold C. Strauss
From The Division of Cardiology, Department of Medicine, and Department of Pharmacology, Duke University Medical Center,
Durham, North Carolina
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SUMMARY. Current theories envision recovery from local anesthetic block of sodium channels
via slow hydrophilic and fast hydrophobic paths. Extracellular pH reduction which increases
cationic/neutral anesthetic form should especially prolong recovery kinetics of highly lipid soluble
compounds that could readily exit via the hydrophobic pathway at normal extracellular pH. To
test this hypothesis, we compared the effects of three related compounds with similar pKa on the
time course of Vmax reactivation in guinea pig papillary muscle at pH<, 7.4 and 6.95. The compounds
were lidocaine and its two desethylation products, monoethylglycinexylidide and glycinexylidide.
Judged from the octanol:water partition coefficient, lidocaine was the most lipid soluble (log
partition coefficient 2.39 ± 0.10), followed by monoethylglycinexylidide (log partition coefficient
1.32 ± 0.09) and glycinexylidide was the least lipid soluble (log partition coefficient 0.41 ± 0.09).
At 30 MM and pHo 7.4, the potency order for Wmix depression at zero diastolic interval was
lidocaine (53 ± 6%), monoethylglycinexylidide (17 ± 3%), and then glycinexylidide (7.8 ± 1.9%).
The decay of V ^ block appeared monoexponential, and the time constant of recovery was dose
independent. Most important is the fact that there were significant differences in the r r increase
with extracellular pH reduction (P < 0.05; Scheffe contrasts). The increase was greatest with
lidocaine [73 ± 28% (mean ± SD)], less with monoethylglycinexylidide (42 ± 15%), and least with
glycinexylidide (13 ± 17%). The simplest interpretation of the differences in extracellular pHdependence of recovery kinetics was that recovery from block due to the neutral form of these
ionizable local anesthetics depended on lipid solubility, whereas recovery from block due to the
protonated form depended on molecular weight. (Circ Res 55: 513-523, 1984)
IONIZABLE antiarrhythmic drugs such as lidocaine
possess the useful property of more potent electrophysiological action in ischemic than in normal myocardium (Kupersmith et al., 1975; Hille, 1978; Hondeghem, 1976; Allen et al., 1978; Lazzara et al.,
1978; Cardinal et al., 1981). This potentiation has
been attributed in part to an increase in H + concentration within ischemic areas (Kupersmith et al.,
1975). Measurements of phase 0 V ^ , reactivation
in guinea pig papillary muscle (Grant et al., 1980)
and sodium conductance reactivation in voltageclamped nerve, skeletal muscle, and Purkinje fibers
(Khodorov et al., 1976; Schwarz et al., 1977; Bean
et al., 1982) indicate that recovery from local anesthetic sodium channel block is delayed by extracellular pH reduction. Acidosis is thought to increase
the proportion of channels occupied by the protonated form of the local anesthetic base (Kupersmith
et al., 1975; Schwarz et al., 1977). Experiments with
permanently charged quaternary amine local anesthetics in nerve and heart indicate that hydrophilic
protonated drug molecules unblock with much
slower kinetics than more lipid-soluble neutral
forms (Strichartz, 1973; Schwarz et al., 1977; Gin-
tant et al., 1983). The kinetic difference has been
attributed to the very large difference in lipid solubility (Hille, 1977; Schwarz et al., 1977; Courtney,
1980). However, studies examining heterogeneous
groups of local anesthetic amines at normal pH have
produced conflicting conclusions as to the importance of lipid solubility in determining recovery
kinetics (Courtney, 1980; Campbell, 1983). If lipid
solubility is of pivotal importance, pH reduction
should especially prolong recovery kinetics of compounds with highly lipid-soluble neutral form, since
these would readily unblock via the membrane
phase at normal pH. We thought it would be of
advantage to restrict the comparison to a small group
of related compounds with different lipid solubility
and similar values for pKa; we therefore chose to
examine the electrophysiological properties of lidocaine and its two desethylation products, monoethylglycinexylidide (MEGX) and glycinexylidide
(GX). Dealkylation is expected to reduce lipid solubility (Hansch and Leo, 1979). We were particularly
interested in determining whether there was a corresponding reduction in the pH dependence of recovery kinetics.
Circulation Research/Vo/. 55, No. 4, October 1984
514
The cellular electrophysiological properties of
MEGX and GX have not previously been described.
They have been shown to accumulate in the plasma
of patients and dogs receiving lidocaine and to partition into the myocardium to an extent similar to
lidocaine (Strong et al., 1973; Halkin et al., 1975;
Handel et al., 1983). MEGX appears to possess antiarrhythmic and convulsant properties comparable
to lidocaine, whereas GX appears considerably less
potent (Smith and Duce, 1971; Blumer et al., 1974;
Tenthorey et al., 1981). We determined lipid solubility by octanol:H2O partition coefficient (P). The
kinetics of recovery from block of phase 0 Vmax were
assessed by determining the time constant of recovery (rr) in guinea pig papillary muscle at normal and
low pH using conventional microelectrodes.
Methods
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Tissue Preparation and Apparatus
Guinea pigs (250-350 g) were stunned and the hearts
rapidly removed and dissected in room temperature-oxygenated Krebs-Henseleit solution. Right ventricular papillary muscles (0.8-1.0 mm in diameter) were mounted in
a three-compartment sucrose gap chamber. The length of
muscle in the test chamber was limited to 0.5-1.0 mm
(Reuter and Scholz, 1968). The 0.7-ml capacity test compartment was perfused at 3 ml/min. Bath temperature was
monitored by thermister (YSI no. 511 probe) and controlled to 36 ± 1°C. The preparation was stimulated with
constant current pulses, 2 msec in duration. Pulse amplitude was that required to maintain constant latency (usually 2 msec) between end of stimulus and peak upstroke
velocity of the action potential. Transmembrane potential
was recorded differentially between two glass microelectrodes containing 3 M KC1 (resistance 15-30 Mfi). In most
experiments, the microelectrode tips were beveled to reduce resistance to 15-20 MQ. One electrode impaled the
cell and the other was positioned in close proximity just
above the tissue surface. The microelectrodes were coupled via Ag/AgCI half-cells (WPI EH-3FS) to matched
voltage followers having high input impedance (1010Q)
and input capacity neutralization. The output of the voltage followers was amplified by the 3A9 differential amplifier of a Tektronix 565 oscilloscope. The maximum rate
of rise of the transmembrane potential (V^*) was obtained
by electronic differentiation (differentiator output linear
over the range 10-1000 V/sec). Oscilloscope traces of the
action potential and its time derivative were photographed
by a Grass C4 camera and enlarged (8x) for analysis by
projection onto a digitizing tablet.
Solutions
Under control conditions, the anterior (test) compartment was perfused with Krebs-Henseleit (K-H) solution
at pH 7.4, containing (ITIM): NaCl, 118; NaHCO3, 21; KC1,
4.3; MgSO4, 1.2; glucose, 11. The solution was gassed
continuously with 95% O2, 5% CO2 starring 10 minutes
before the addition of CaCh. To test the effects of acidosis,
we reduced pH to 6.95 by replacing all but 7.5 rrtM
NaHCO3 with NaCl. The pH of the solution was titrated
to 7.4 or 6.95 by the addition of HC1 or NaOH, using a
high precision pH analyzer (IL Inc., model 113). Preliminary experiments showed that solution pH remained constant from solution reservoir to anterior chamber; we
routinely monitored reservoir pH, not anterior chamber
pH, to avoid disturbing the preparation. Acidosis increases
ionized calcium activity (Spitzer and Hogan, 1979). The
present pH reduction increased the ionized calcium activity of our K-H solution from 1.41 ± 0.01 to 1.57 ± 0.01
mM (mean ± SD, n = 3). We decided not to reduce the
calcium concentration of the pH 6.95 solutions to counteract this change, since no such compensation would
occur in vivo.
The middle compartment was perfused with isotonic
sucrose solution containing 0.01 mM CaCl2 and gassed
with 100% O2. The posterior (current-injecting) compartment was perfused with isotonic KC1 solution produced
by substitution of all NaCl in the standard K-H solution.
Procedures
An initial 60-minute equilibration period in control
solution was allowed. Once a stable impalement had been
obtained, action potential characteristics and Vmax recovery
kinetics were recorded. Recordings were repeated after 30,
60, and 120 minutes of exposure to lidocaine, MEGX, and
GX, respectively. Only one drug was tested in each preparation. The metabolites, being less lipid soluble, required
longer exposure times to produce a stable effect than did
lidocaine. The basic drive stimulus (SI) was delivered in
repetitive 10-beat trains at 1 Hz separated by 2-second
pauses. By definition, diastole began when phase 3 repolarization had returned the membrane potential to 1 mV
from its resting value. To determine recovery kinetics, a
test stimulus (S2) was introduced at varying diastolic
intervals following successive trains. Any reduction in Vmax
of the test action potential was expressed as a fraction of
the amplitude during the preceding basic drive action
potential according to the expression: fractional Vmax reduction, (b) = (VmaxSi - VmaxS2)/Vmai<si. Since Vmax reactivation followed a single exponential in the present experiments, the decay of b from its value at zero diastolic
interval (b0) is given by b(t) = b0 exp (—t/r,), where T, is
the time constant of reactivation. Thus b decays linearly
over time in a semilogarithmic plot. After logarithmic
transformation of b, linear regression techniques were
used to obtain least squares estimates of b0 and the slope
(regression coefficient) of the decay. rr is simply the inverse slope.
It was easier to measure the kinetics of recovery from
lidocaine block of Vmax than metabolite block, because of
lidocaine's greater potency. With GX, the least potent, it
was necessary to average =5 replications at each diastolic
interval to obtain a sufficiently noise-free estimate of the
time course of Vmax reactivation. This approach was used
in all GX experiments and some MEGX experiments. However, our criteria for adequate kinetics determination (see
Statistical Analysis) could be satisfied for MEGX and
lidocaine without the use of replication.
In some preparations, the effects of two concentrations
of a single drug were examined (15 and 30 /IM lidocaine
or MEGX; 30 and 120 //M GX). To test the effects of low
HCO3 acidosis in drug-free solution and during exposure
to drug, the sequence of pH 7.4 and 6.95 solutions was:
pH 7.4 (60 minutes), pH 6.95 (20 minutes), pH 7.4 (20
minutes), pH 7.4 with drug (30, 60, or 120 minutes for
lidocaine, MEGX or GX, respectively), pH 6.95 with drug
(20 minutes), pH 7.4 with drug again (60 minutes). Because of the difficulty in maintaining long impalements,
it was often not possible to record the entire sequence
within the one cell. Partial experiments are reported pro-
515
Broughton et al./pH, Lipid Solubility, and Vmax Reactivation Block
vided data were collected from the same cell before and
during the particular intervention being examined. That
is, a return to pre-intervention conditions was not always
possible.
Octanol:H2O Partitioning
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Differences in lipid solubility between lidocaine and its
two metabolites were assessed by comparison of octanol:water partition coefficients. Partitioning was examined 2 pH units above, and 2 pH units below, drug pKa to
reflect properties of neutral and charged drug molecules,
respectively. Siliconized 15-ml glass test tubes containing
cross-saturated octanol (2 ml) and water (2 ml) were gently
rocked for 1 hour at 23°C after the addition of drug. The
water was pipetted off after centrifugation at 1500 rpm
for 5 minutes. Initial aqueous phase drug concentration
(=167 Mg/ml) and final aqueous concentration after partitioning were measured by reverse phase ion-pair HPLC
(Laboratory Data Control, model 1204A; 4.6 mm X 25
cm, Zorbax ODS column). The mobile phase contained
4.52 g NaH2PO4, 10 ml triethylamine (Aldrich), and 130
ml acetonitrile (Fisher Scientific, HPLC grade). Mobile
phase pH was lowered to 3.0 with phosphoric acid before
the addition of acetonitrile. The mobile phase was pumped
at 1.9 ml/min, and the UV detector was set to measure
absorbance at 200 nm. Estimates of drug concentration
were the average of five replications.
Statistical Analysis
Student's f-test for paired data was used to assess the
significance of pairwise comparisons within cells (resting
membrane potential before and during drug exposure, or
at two different pH values, etc.). The value during an
experimental intervention was compared to the average
of the value before and the value following recovery if
both were available—otherwise, to the value beforehand.
To compensate for simultaneous multiple comparisons,
for example, when assessing the effect of GX 30 /xM on
resting potential, action potential duration and steady state
Vma», a method analogous to the Bonferroni method, was
used (Wallenstein et al., 1980). The probability value
corresponding to the non-modified f-statistic for the difference between each pair of means was found to three
significant figures by linear interpolation using logarithms
of the standard two-tailed probability values (Rohlf and
Sokal, 1969). A significance level of P < 0.05/m was used,
where m = number of simultaneous comparisons. P values
are reported together with their correction factor when
derived from paired f-tests (e.g., P < 0.05/3).
One-way analysis of variance was used to assess the
significance of differences in recovery kinetics across the
three drugs, since the data were obtained in separate cell
populations. Scheffe's method was used to correct for the
multiple pairwise comparisons among lidocaine and the
two metabolites (Wallenstein et al., 1980).
Experimental noise sometimes prevented reliable estimates of recovery kinetics being obtained. Linear regression techniques were used to eliminate from analysis
experiments with unacceptable variance of the In b —
diastolic interval relation [correlation coefficient <0.9, or
regression coefficient not significant at the 1 % level (Snedecor and Cochran, 1967)]. Regression analysis was also
used to assess the significance of any change in T,, since
this reflected a change in regression coefficient. Here, we
obtained the F ratio of the residual mean square variance
when one slope, as opposed to two slopes, was fitted to
the data (Snedecor and Cochran, 1967).
Results
Steady-State Action Potential Characteristics
As expected at pH,, 7.4,15 nu lidocaine shortened
action potential duration (P < 0.05/3; Table 1).
However, resting potential and steady-state V^x
remained closely similar to the lidocaine-free values
at the stimulation rate of 1 Hz, consistent with
previous reports (see Chen et al., 1975). Neither 15
tiM MEGX nor 30 MM GX had any effect on these
three variables. No further changes were observed
after doubling the dose of lidocaine (n = 6) and
MEGX (n = 7) or quadrupling the dose of GX (n =
3). Reducing pHo to 6.95 increased action potential
duration significantly, both under drug-free conditions and during exposure to lidocaine and MEGX
(P < 0.01/3; Table 2). A similar increase with acidosis during exposure to GX approached, but did
not reach, statistical significance (3P = 0.062). pH<,
reduction produced a small decline in steady-state
Vmax which was significant with lidocaine (—11%)
and MEGX (-14%), but not under drug-free conditions or GX exposure (Table 2). pHo reduction also
produced a small reduction in resting potential in
the absence and presence of drug, but the change
was statistically significant only when drug-free and
drug results were pooled (1.4 mV, P < 0.01/3, n =
26).
V max Reactivation
Drug-Free Conditions
At pHo 7.4, Vmax recovery was virtually complete
(>98%) by the time membrane potential had repolarized to 1 mV from its resting value. The subsequent small residual recovery approached experimental noise. In 25 muscles, control time constants
of Vmax reactivation were not calculable in 23 (<20
msec). In the remaining two muscles, the values
were 26 and 36 msec. pHo reduction to 6.95 had
little effect on reactivation kinetics (« = 8). Recovery
may have been slightly prolonged (e.g., Fig. 1, panel
TABLE 1
Effect of Lidocaine, MEGX, and GX on Action Potential
Characteristics at 1 Hz in Guinea Pig Papillary Muscle
RMP
(mV)
APD
(msec)
(V/sec)
11
85.8 ± 6 . 3
+0.3
238 ± 26
-9*
224 + 51
-3
Control
MEGX (30
6
85.0 ± 1 .1
+1.0
197 ± 21
-2
285 + 44
-8
Control
5
82.9 ± 0 .6
-0.8
203 ± 27
+4
234 + 25
-16
Control
Lidocaine (15 ,IM)
GX (30 jiM]1
Results are mean ± SD in control solution, average change in
absolute units with drug. RMP, resting membrane potential; APD,
action potential duration until repolarization to 1 mV from RMP;
Vouu, maximum upstroke velocity of action potential.
* P < 0.05/3 for difference from control.
516
Circulation Research/Vol. 55, No. 4, October 1984
TABLE 2
Effect of Reducing Extracellular pH on Action Potential Characteristics at 1 Hz under Drug-Free
Conditions and after Lidocaine, MEGX and GX
RMP
(mV)
APD
(msec)
(V/sec)
8
85.6 ± 2.6
-1.1
208 ± 31
+19*
227 ± 28
-13
7.40
6.95
6
83.3 ± 2.5
-2.1
219 ± 20
+24
209 ± 39
-22*
7.40
6.95
6
82.9 + 4.5
-1.3
208 + 21
+ 19*
262 + 38
7.40
6.95
6
82.7 ± 1.5
-0.9
195 + 25
+25
222 ± 51
-14
pH
n
Drug free
7.40
6.95
Lidocaine (15 HM)
MEGX (30 ^M)
GX (30
JJM)
-36f
Results are means ± SD at pH 7.40, average change in absolute units at pH 6.95. Abbreviations as
in Table 1.
* P < 0.01/3; t P < 0.05/3 for difference between the values at each pH.
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3) but could not be quantified in five of eight muscles
(Fig. 1, panels 1 and 2).
MM (b0 = 0.43 ± 0.08, n = 12), MEGX 30 »M (b0 =
0.17 ± 0.03, n = 10), and then GX 30 m (b0 = 0.078
± 0.019, n = 10). These differences were significant
at the 1% level (one-way analysis of variance;
Scheffe contrasts). As can be seen from Figure 2,
unblocking following the train on average proceeded most rapidly with lidocaine (P < 0.05), then
with GX, and least rapidly with MEGX (P < 0.01).
An increase in dose of the three compounds increased b0 but did not alter the kinetics of Vmax
reactivation (Fig. 3; Table 3). To determine how
closely recovery from lidocaine block approximated
Drug Exposure
At pHo 7.4, Vmax recovery was delayed by exposure to both metabolites, as well as to lidocaine
itself. There were marked differences among the
three compounds in the level of Vmax block present
at the beginning of diastole, and moderate differences in the subsequent time course of unblocking
(Fig. 2). The order of potency for fractional V ^
block at zero diastolic interval (b0) was lidocaine 15
1.0
DRUG-FREE
DRUG-FREE
DRUG-FREE
b
D
8
O
MUSCLE
DEOl
MUSCLE
15D
O
0.01
-50
0
50
100 -50
0
50
100 -50
DIASTOLIC INTERVAL (ms)
0
50
100
FIGURE 1. Three individual examples of the time course of Vm*, reactivation under drug-free conditions at extracellular pH 7.4 (filled circles) and
6.95 (empty circles). Data are expressed as fractional Vmo reduction (b) in the "test" vs. "basic drive" action potential (1 Hz) as described in the
legend to Figure 4. Data points falling prior to zero diastolic interval (see Methods) were excluded from analysis. The time constant of Vma
reactivation (T,) was too short to be calculated from later points in muscles DEOl and 15D at either pH. T, was slightly prolonged by acidosis in
muscle MG17. The lines through the data points in the right panel were fitted by the method of least squares from the regression of In b on
diastolic interval.
Broughton et a/./pH, Lipid Solubility, and Vmol Reactivation Block
1.0
LIDOCAINE I
n
Tr
I39*26rm 12
MEGX 30^.M
232*34
10
GX 30/i.M
182t35
10
b o.i
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0.01 0
250
500
DIASTOLIC INTERVAL (ms)
FIGURE 2. Average time course of Vmal reactivation at pH0 7-4 during
exposure to lidocaine, MEGX, or GX. The slope and elevation of each
line are means of the slopes and elevations at zero diastolic interval
(bo) obtained in individual muscles from the regression of In b on
diastolic interval (see legend to Figure 4). Vertical bars on left show
variance (±1 SD) around the mean b0 for each compound.
a monoexponential decay, recovery kinetics were
determined particularly in an additional 18 muscles.
The Vmax measurements at each coupling interval
were averaged over five replications to minimize
variance from experimental noise. The relationship
between In b and time was linear in all cases, with
the correlation coefficient averaging 0.990 (range
0.942-0.999) in the 18 experiments. Thus, the decay
in Vmax block appeared monoexponential (Fig. 4).
517
Reduction in pHo to 6.95 markedly delayed the
recovery from lidocaine block, as expected (Figs. 5
and 6). The kinetics of recovery from MEGX block
were also slowed significantly (P < 0.01), but to a
lesser extent (P < 0.05). By contrast, recovery from
GX block was not significantly affected by pH<,
reduction (Figs. 5 and 6). The increase in recovery
time constant produced by acidosis averaged 73 ±
28% with lidocaine (P < 0.01; n = 6), 42 ± 15%
with MEGX (P < 0.01; n = 9), but only 13 ± 17%
with GX (P > 0.4; n = 7). The change in recovery
kinetics in each individual muscle was tested by
linear regression analysis. The change in slope of
the linear relationship between In b and diastolic
interval was significant in all six muscles exposed to
lidocaine (P < 0.001 in each), in eight of nine
muscles exposed to MEGX (P < 0.025 or better), but
in only one of seven musles exposed to GX (P <
0.05 in this muscle).
Acidosis also reduced b 0 due to MEGX (from 0.17
± 0.036 to 0.121 ± 0.028, P < 0.01). No comparable
effect was observed with lidocaine or GX (Fig. 6).
Octanol:H2O Partitioning
Considerable differences in lipid solubility of the
neutral form were suggested by the partitioning
differences among the three compounds in vitro at
pH = pKa +2. Lidocaine was the most lipid soluble.
P, the ratio of octanol:H2O concentrations after partitioning, was 249 ± 60 (« = 4). MEGX was next
most lipid soluble, with the ratio of octanol:H2O
concentrations averaging 21.2 ± 4.3 (n = 3). GX was
the least lipid soluble (P = 2.56 ± 0.3, n = 3).
Biological activity has been found to vary with log
lipid solubility (Hansch and Leo, 1979; Courtney,
1980). The differences in log P are shown in Table
4. The value for lidocaine is almost twice that of
MEGX and almost six times that of GX. Partitioning
under conditions pH = pKa — 2 excluded measurable
GX
MEGX
LIDOCAINE
Tr
"o
120/i.M. U 5 r m .14
.077
3 0 F M . 159
120flM
30/iM
MUSCLE AGI5
0
250
500 0
250
500 0
DIASTOLIC INTERVAL (ms)
250
500
FIGURE 3. Individual examples of Vma reactivation during exposure to two different concentrations of lidocaine, MEGX, or GX. Methods and
abbreviations as in legend to Figure 4, except that data points in the left and middle panels represent one test action potential at each diastolic
interval, not the mean of five replications.
Circulation Research/Vol. 55, No. 4, October 1984
518
TABLE 3
Recovery Kinetics from V^, Block at Two Doses of Lidocaine, MEGX, and GX in Guinea Pig
Papillary Muscle at 1 Hz
Lower dose
Higher dose
T,
Lidocaine (15 and 30 MM)
MEGX (15 and 30 MM)
GX (30 and 120 MM)
6
5
3
b0
(msec)
b0
(msec)
0.385 + 0.024
0.106 ±0.014
0.065 ± 0.010
143 ± 32
228 ± 45
180 ± 28
0.534 ± 0.058
0.182 + 0.027
0.145 ± 0.032
146 ± 27
226 + 45
175 ± 28
Data are means ± SD; b0, fractional Vmsx block at the onset of diastole; T,, time constant for decay of
V™ block.
lipid solubility of the charged (protonated) form of
lidocaine, MEGX, and GX.
Discussion
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In the doses examined, neither lidocaine nor its
desethylated metabolites, MEGX and GX, depressed
phase 0 Vmax of the 1 Hz basic drive action potentials.
However, test impulses detected significant Vmax
block in early diastole following a 1 Hz train in the
1.0 r
LIDOCAINE 15/iM
r = 0.999
slope = 0.0064910.000085
T> = 154 ms |P<0.001)
b
MUSCLE J22
\ DRUG FREE
Q X, 36 mj
0.01
LA
0
250
500
DIASTOLIC INTERVAL (ms)
FIGURE 4. An individual example of lidocaine's effect on the time
course of Vm^ reactivation in guinea pig papillary muscle at pH0 7.4.
Reactivation was assessed by varying the coupling interval of a single
"test" action potential following a 1 Hz train. Data show the timedependent decay in fractional Vma block (b), where b = [Vmax (1 Hz
train) — Vma (test)]/]/^ (1 Hz train). Each data point is the average
of five replications. The lines through the data points were fitted by
the method of least squares from the regression of In b on diastolic
interval. The correlation coefficient (r) shows the data is well fitted
by a straight line. Calculated b at zero diastolic interval (bo) was 0.52
in this muscle. The significance of the slope value (regression coefficient) was obtained from "t" = the ratio of slope to its standard
deviation (degrees of freedom n-2; Snedecor and Cochran, 1967). The
time constant of Vmia reactivation (rr) is the inverse slope.
presence of each of the three compounds. At equimolar concentration, the initial diastolic block (b0)
was most intense with lidocaine, of lesser intensity
with MEGX, and least with GX. At 30 /IM, b 0 averaged 0.53 with lidocaine (Table 3), 0.17-0.18 with
MEGX (Fig. 2; Table 3), but only 0.07-0.08 with GX
(Fig. 2; Table 3). These differences roughly paralleled the relative lipid solubilities of the three compounds, which presumably determined the membrane concentration of neutral anesthetic molecules
adjacent to sodium channels (relative log P
1.0:0.55:0.17, for lidocaine, MEGX, and GX, respectively). It would be necessary to obtain full doseresponse curves to determine how closely potency
for early diastolic block is a function of lipid solubility as assessed from octanol:H2O partitioning.
All three compounds slowed the kinetics of Vmax
reactivation. Significant differences were observed
in the rate of diastolic decay of Vmax block. At pHo
7.4, lidocaine block dissipated most rapidly (time
constant r r 139 msec), followed by GX (rr 182 msec),
whereas MEGX unblocked most slowly (rr 232 msec)
(Fig. 2). These differences were not a consequence
of the potency differences for b0 block. Reactivation
kinetics were shown to be dose (and hence b0)
independent for the three compounds over the range
of b 0 values from 0.08 to 0.53 (see Fig. 3 and Table
3).
The question then arises whether the differences
in Vmax reactivation can be taken to indicate comparable differences in the rates of sodium channel
unblocking among the three compounds. Although
a close correlation between Vmax and peak inward
sodium (INa) current is expected in cardiac muscle
on theoretical grounds, whether Vmax is a reliable
index of available sodium conductance (gNa) is controversial (Cohen and Strichartz, 1977; Hondeghem,
1978; Strichartz and Cohen, 1978; Walton and Fozzard, 1979; Cohen et al., 1981; Bean et al., 1982).
Bean et al. (1982) have recently reported that, despite the precaution of constant latency, Vmax appeared to be a nonlinear index of gNa changes with
tetrodotoxin in voltage clamp experiments. A similar
nonlinear relationship between Vmax and gNa was
obtained when the number of sodium channels
available to open during a sudden depolarization
was varied by using different holding potentials to
Broughton et al./pH, Lipid Solubility, and Vm<lr Reactivation Block
10
r
LIDOCAINE 15>iM
f
519
GX 30/iM
MEGX 30/iM
pH
Tr
6.95
7.4
235 mi
163
pH
.12
.15
6.95
7.4
T,
162«
130
bo
.086
.093
b 0.1
MUSCLE DEOI
0.01
500 0
250
250
500 0
DIASTOLIC INTERVAL (ms)
500
250
Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017
FIGURE 5. Individual examples of the effect of extracellular pH reduction on Vma reactivation during exposure to lidocaine, MEGX, or GX.
Abbreviations and method for determining recovery kinetics as in legend to Figure 4, except that data points in the left panel represent one test
action potential at each diastolic interval, not the mean of five replications.
change inactivation (Cohen et al., 1984). An important consequence predicted from their study was
that the time course of reactivation measured by
Vmax will be distorted and nonexponential (curvilinear) when the underlying recovery of gNa is exponential. In the present study at 37°C, we did not
observe curvilinear Vmax reactivation. The kinetics
we obtained closely approached monoexponential
decay except for a slight deviation at the shortest
coupling intervals consistent with recovery of drugfree channels (Fig. 4).
Clarification of the discrepancy between the two
methods is impeded by the current difficulty of
obtaining reliable measurements of sodium current
in heart under the same conditions of physiological
sodium concentration, membrane potential and temperature at which Vmax measurements are obtained.
1.0
LIDOCAINE 15^M(n=6)
Effects of pH, Reduction
The kinetics of recovery from lidocaine block of
Vmax was markedly slowed by extracellular acidosis,
MEGX 30/iM(n = 9
pH
T,
I
6.95 231J26mi .47*07
7.4 !36t20 .47t.O9
b
Nonetheless, Vmax provides an indirect measure of
sodium conductance which must be interpreted with
great caution. Linear kinetics of Vmax reactivation are
a necessary but not sufficient feature ensuring proportionality with gNa reactivation. Complex interactions of activation and inactivation gating processes
producing an apparently pure semilogarithmic decay but with an incorrect slope cannot be excluded
(see Bean et al., 1982). However the b 0 independence
of Vmax reactivation probably ensures that directional
changes in the time course of sodium channel reactivation is reliably indicated.
pH
Tr
6.95 3l4*57n» I2J.O3
7.4 22U27
.I7S.04
GX 30/xM (n = 7)
pH
T,
6.95
2 0 I * } 4 I « I .078! 033
7.4
185:43
.084!.019
PH6.95
pH7.4
0.01
0
250
500 0
250
500 0
DIASTOLIC INTERVAL . ms
250
500
FIGURE 6. Average time course of V^ reactivation at pHo 7.4 and 6.95 during exposure to lidocaine, MEGX, or GX. The slope and elevation of
each line are the means of the slopes and elevations at zero diastolic interval (bo) obtained in individual muscles from the regression of In b on
diastolic interval (see legend to Figure 4).
520
Circulation Research/Vol. 55, No. 4, October 1984
TABLE 4
Physicochemical Properties of Lidocaine, MEGX, and GX
[Neutral]/[protonated]
Lidocaine
MEGX
GX
Molecular wt
234
206
178
pK.
pH.7.4
pH, 6.95
logP*
7.86t
8.04J
7.68J
0.35
0.23
0.53
0.12
0.081
0.19
2.39 ± 0.20 (n = 4)
1.32 ± 0.09 (« = 3)
0.41 ± 0.06 (n = 3)
* Octanol:H2O partition coefficient at pH = pK« + 2; five replicates each estimation,
t Truant and Takman, 1969; J Johansson, 1982.
Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017
as previously reported by Grant et al. (1980). Interestingly, recovery from MEGX block was less affected, and recovery from GX block almost completely unaffected, by moderate pH,, reduction. Current theories attribute the exponential recovery of
sodium conductance between action potentials to
drug exiting from sodium channels (Strichartz, 1973;
Courtney, 1975; Hille, 1977, 1978; Hondeghem and
Katzung, 1977). Hydrophobic neutral drug molecules are thought to return rapidly to the membrane
phase once transmembrane potential no longer favors channel occupancy; the lipid-insoluble charged
form must exit via the hydrophilic channel inner
mouth unless it loses its charge (Hille, 1977). Channel-gating configuration in diastole is thought to
impede escape of the charged species from the
blocking position, although other explanations for
slow unblocking kinetics have not been excluded.
Reduction in pHo is thought to favor protonation of
drug molecules within the channel to an extent
dependent on drug pKa. Lidocaine, MEGX, and GX
are weak bases with pKa 7.86, 8.04, and 7.68, respectively. The protonated form predominates at
low pH o . However, in the present study, the alteration in recovery kinetics with pHo reduction did
not correlate with the calculated change in ratio of
neutral to protonated form (Table 4). Indeed, the
calculated change was greatest with GX, which
showed little or no potentiation at all. This apparent
contradiction is resolved if an important role is proposed for lipid solubility in determining the unblocking rate of the neutral form (Hille, 1977; Courtney,
1980). Thus, an increase in protonated:neutral form
should especially prolong recovery kinetics of a
highly lipid soluble compound like lidocaine, which
could readily exit via the membrane phase at normal
pH. By contrast, lipid-insoluble GX unblocked relatively slowly whether protonated or not. MEGX with
intermediate lipid solubility of the neutral form
showed intermediate pH potentiation as far as kinetics of unblocking are concerned.
The question arises as to whether the marked
slowing of lidocaine unblocking that we observed
can reasonably be attributed to the modest reduction
in ratio of neutral:protonated form produced with
reduction in pHo to 6.95. To clarify this question,
we examined two simple kinetic alternatives. The
binding of neutral and protonated drug (Dn,Dc) to a
drug-free channel (U) to form a blocked channel
(Bn,Bc) was restricted to the duration of the action
potential and was assumed to be negligibly slow at
the present micromolar concentrations once repolarization was complete.
Scheme 1 (uncoupled)
U + Dc ^ Bc
U + Dn
Scheme 2 (coupled)
U
Bn
Dc ^
Bc
U + Dn ^
Bn
where k is a composite rate constant term describing
drug association during diastole which combines
rate contants for binding, diffusion, field effect, and
drug concentration; / is a composite rate constant
term describing drug dissociation during diastole
which combines rate constants for binding, diffusion, and field effect; and where /„ » kn and 4 ^>
kc in schemes 1 and 2 during diastole (Strauss et al.,
in press).
Fractional block at the onset of diastole (b0)
equaled the sum of block due to both neutral and
charged species (bn + bc). In scheme 1, the fraction
of charged and neutrally blocked channels (bn and
bc) decay as independent processes. In other words,
there is no conversion of Bc to Bn, governed by pH o .
In scheme 2, interconversion between charged and
neutrally blocked channels is permitted to maintain
the value of R (bn/bc) constant throughout the decay
in total Vmax block. We assume here that ambient
pH and drug pKa within a blocked channel are the
same as for drug in extracellular solution, so that R
= b n /b c = IOP^-P"*. The coupled scheme was
thought more realistic in view of the known access
of extracellular protons to the channel-binding site
(Schwarz et al., 1977) and the high speed of proton
transfer reactions (Bell, 1973). As can be seen from
Figure 7, scheme 1 is unlikely to account for the pH
potentiation we observed with lidocaine. Time constants for the exponential decay of neutral and protonated species block (r n , TC) were assumed to be 50
and 500 msec, respectively. The decay in total block
is biexponential with an early rapid phase due to
Broughton et fl/./pH, Lipid Solubility, and V™, Reactivation Block
1.0
AGGREGATE DECAYS
pH: 7.4
6.95
b 0.1
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0.01
0
250
500
DIASTOLIC INTERVAL (ms)
FIGURE 7. Simulated effect of extracellular pH reduction on Vmol
reactivation according to kinetic scheme 1 (see Discussion). It is
assumed that recovery from Vmal block due to protonated lidocaine
(LH+) and recovery from block due to neutral lidocaine (L) occur as
two independent monoexponential decays with time constants 500
and 50 msec, respectively. At zero diastolic interval, bn:bc, the ratio
of block due to L and block due to LH+, is assumed to equal the ratio
[L]:[LH+] in the extracellular fluid. That is, bjbc = W"''1"^. The
curvilinear aggregate decay in total b from a b0 value of 0.5 at each
pH was found by addition of the linear decays of bn and bc.
recovery from neutral species block and a subsequent slow phase due to recovery from protonated
species block. Our lidocaine data were much better
fit by scheme 2 which, for r n <3C TC, as above, implies
that a significant proportion of the decay in bc
involves a hydrophobic pathway of drug egress as
molecules lose their charge and exit via the membrane phase. For scheme 2, where b n /b c remains
constant throughout diastole, bn = Rb/(1 + R) and
bc = b/(l + R). If /„ and fc are rate constants for
decay of b n and b c , respectively, then:
521
rate constants for neutral and charged forms of
lidocaine correspond to r n and TC values of 44 and
483 msec, respectively, at the resting potential of
the present experiments (—82 mV). The predicted
relationship between r r and R from Equation 2,
assuming the above values for /„ and fc for lidocaine,
is shown in Figure 8. It can be seen that recovery
kinetics of V^,* block approaches the TC value as R
decreases, particularly once R is less than 0.3. This
would correspond to pHo at least 0.5 U below pKa.
Schwarz et al. (1977) observed even more steep pH
dependence of recovery from lidocaine block of INa
in frog skeletal muscle at 12.5°C.
For MEGX, the r n and TC values were 70 and 439
msec, respectively. For GX, r n was 144 msec and TC
was 217 msec. The simplest interpretation of these
differences is that rn decreases as lipid solubility
increases, whereas r c decreases with molecular
weight. However, the relation between physicochemical properties and kinetics of drug action is
complex (Campbell, 1983), and it has yet to be
demonstrated that extrapolation of these conclusions beyond this small series of related low molecular weight compounds is valid. The analysis predicts unexpectedly fast recovery kinetics for the
protonated tertiary amine species in view of results
obtained with permanently charged quaternary
compounds (Gintant et al., 1983). The discrepancy
may reflect steric hindrance from the more bulky
amine region of the quaternary molecule. Bean et al.
(1983) recently published data on the time course of
sodium current reactivation in rabbit Purkinje fiber
exposed to lidocaine under voltage clamp at pHo 7.0
500
400
300
AGGREGATE
(ms)
200 -
db/dt = -/ n Rb/(l + R) - / c b/(l + R)
Thus
b(t) = b 0 exp - (/nR
R)
(1)
Thus, reactivation should be monoexponential with
the time constant for decay in total V ^ block (rr)
given by
R)/(/nR
(2)
Knowing our experimental values for r r at pH, 7.4
and 6.95, and calculating R, Equation 2 can be
solved for / n and 4 giving values of 2.25 X 10"2 and
2.07 x 10~3 msec"1, respectively. These dissociation
(RATIO OF NEUTRAL TO CHARGED DRUG)
FIGURE 8. Predicted relationship between the time constant of Vmla
reactivation (aggregate T,) and the ratio of neutral to protonated
lidocaine, MEGX or GX during changes in extracellular pH, according
to kinetic scheme 2 (see Discussion). It is assumed that bn:bc, the ratio
of Vm4t block due to neutral drug-.block due to protonated drug within
sodium channels, remains identical to the value ofR, the ratio [neutral
drug]:[protonated drug] in the extracellular fluid, throughout the
decay in total b. R= 10'"'''*:
522
and 8.1. Application of our kinetic analysis to their
data also predicts relatively rapid unblocking of the
protonated form of lidocaine (TC 1000 msec), but
leads to a surprisingly large value for the time constant of the lipid-soluble neutral form of the molecule (r n 342 msec). This may reflect maintenance of
their preparation at a temperature below the point
of phase transition of membrane lipids.
Further studies are required to validate the relationship between the aggregate r r and R over a wider
range of R, as the estimates of dissociation rate
constants for the charged and neutral forms of the
compounds are sensitive to the accuracy with which
the decrease in Vmax measures sodium channel block.
Additional studies are also needed to confirm the
importance of lipid solubility for pH potentiation of
local anesthetic block.
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Clinical Implications
Serum levels of MEGX and GX approaching or
exceeding 20 JIM have been reported in human subjects receiving lidocaine infusion. Previous experimental studies suggest that this MEGX concentration
would produce some antiarrhythmic effect. MEGX
was reported to have about one-third the equimolar
potency of lidocaine for antifibrillatory action in
chloroform-exposed mice, one-half the potency for
premature ventricular contraction suppression in
dogs after coronary ligation, full potency for
suppression of ouabain-toxic atrial arrhythmias in
guinea pigs, and one-fourth the potency for block
of frog sciatic nerve (Ehrenberg, 1948; Smith and
Duce, 1971; Burney et al., 1974). In comparison, we
found that the level of Vmax block immediately after
action potential repolarization was one-third of the
block with lidocaine at 30 tiu concentration, similar
to the reported potency ratios for nerve block and
suppression of ventricular arrythmia. In addition,
the kinetics of recovery from MEGX block was prolonged somewhat by acidosis, which may promote
suppression of arrhythmia in ischemic myocardium.
GX was reported one-fourth as potent as lidocaine
in a model of ventricular arrhythmia (Tenthorey et
al., 1981), but failed to suppress ouabain-toxic atrial
arrhythmias (Burney et al., 1974). The present study
suggests that its antiarrhythmic action probably is
minimal at clinically observed blood levels in patients with ischemic ventricular arrhythmias, since
Vmax block at the onset of diastole was less than onesixth that seen with lidocaine, and recovery from
block was not delayed by acidosis.
The work could not have been carried out without the generous
support of Dr. J.C Greenfield, Jr. We are grateful to Astra Pharmaceutical Products, Inc., Worcester, MA for supplies of lidocaine,
MEGX, and GX. Mali Hutchison rendered invaluable technical expertise.
Supported by National Institutes of Health Grants 19216 and
17670. Part of the work was carried out while Dr. Broughton was an
Overseas Research Fellow of the National Heart Foundation of Australia.
Circulation Research/Vol. 55, No. 4, October 1984
Dr. Broughton's present address: Baker Medical Research Institute,
Commercial Road, Prahran, Victoria, Australia 3181.
Address for reprints: Harold C Strauss M.D., Box 3845, Duke
University Medical Center, Durham, North Carolina 27710.
Received October 31, 1983; accepted for publication August 2,
1984.
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INDEX TERMS: Lidocaine metabolites •
block • V~,, reactivation kinetics • Acidosis
Local
anesthetic
Lipid solubility modulates pH potentiation of local anesthetic block of Vmax reactivation in
guinea pig myocardium.
A Broughton, A O Grant, C F Starmer, J K Klinger, B S Stambler and H C Strauss
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Circ Res. 1984;55:513-523
doi: 10.1161/01.RES.55.4.513
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