771
Basis of pH-Independent Inhibitory Effects
of Lactate on 45Ca Movements and Responses
to KCl and PGF2tt in Canine Coronary
Arteries
R. KELLY HESTER, GEORGE B. WEISS, AND JAMES T. WILLERSON
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SUMMARY Under ischemic conditions, contractile responses of canine coronary artery smooth
muscle are decreased, pH is decreased, and lactate levels are increased. To investigate whether lactate
has a pH-independent relaxant action on contractility, inhibitory effects of 1.0 mM lactate on 46Ca fluxes
and increased tension elicited with either 60 mM KCl or 10 ftyi prostaglandin F2o (PGF2o) were measured
at physiological pH (7.4) or in the presence of lactic acidosis (pH 6.9-7.0). In addition to isometric
tension responses, 45Ca exchangeability and total 45Ca uptake and efflux, "Ca retention at nonsuperficial
sites or stores was measured after incubation with an isosraotic (80.8 mM) La3* solution at 0.5 °C.
Pretreatment with lactate specifically depressed PGF2O-induced tension responses more than KC1induced ones, but Ca2+ depletion inhibited the response to KCl more rapidly. The rate of efflux of "Ca
from coronary arteries into a calcium-free solution was decreased by lactate; this effect was not
prevented by inhibition of "Ca reuptake and rebinding with 0.05 mM EDTA. Exchangeability of "Ca
with non-radioactive Ca2+ was decreased by lactate. A Scatchard plot of Ca2+ uptake in coronary
arteries shows that Ca2+ is bound at either high or low affinity sites. Lactate increased the rate of "Ca
uptake at high affinity sites but did not increase the equilibrated total 45Ca uptake. However, the
retention of 45Ca (residual Ca2+ uptake) at high affinity sites was increased by lactate and decreased by
PGF2,,, whereas low affinity residual Ca2+ uptake was increased by KCl and unaffected by PGF2<, or
lactate (although lactate inhibited the KCl-induced increase in low affinity residual Ca2+). Thus, lactate
acts directly to increase the affinity for Ca2+ at high affinity Ca2+-binding sites important for the
stimulatory action of PGF2l>, which appears to increase tension by mobilizing Ca2* from these sites, and
only indirectly and to a lesser degree affects that low affinity Ca2+ important for the stimulatory action
of KCl. CircRes46: 771-779, 1980
INCREASES in lactate concentration, concomitant
with changes in pH, occur with ischemia in striated
and smooth muscles. Decreases in pH associated
with increases in lactate levels have been correlated
with direct depression of the contractile process, as
well as with possible effects on transmembrane Ca2+
fluxes in vascular smooth muscle (Carrier et al.,
1964; Mrwa et al., 1974; Peiper et al., 1976; Turnheim et al., 1977). Conversely, a direct effect of
lactate independent of changes in pH recently has
been demonstrated in cardiac muscle (Crie et al.,
1976; Marrannes et al., 1979). Lactate also prevents
the mannitol-induced increase in contractility of
isolated cat papillary muscles (Crie et al., 1976) in
From the Departments of Pharmacology and Internal Medicine (Cardiovascular Division), University of Texas Health Science Center at
Dallas, Dallas, Texas.
This work was supported in part by the American Heart Association
(Texas Affiliate), National Institutes of Health Grant HL-14775 and
National Institutes of Health Ischemic Specialized Center of Research
HL-17669.
The present address for Dr. Hester is: Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University,
College Station, Texas 77843.
Address for reprints: Dr. George B. Weiss, Department of Pharmacology, University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
Received October 8, 1979; accepted for publication January 29, 1980.
a pH-independent manner. Conduction velocity in
isolated rabbit atria also is depressed by lactate at
normal pH, but this effect is much more dramatic
at a decreased pH of 6.8 (Marrannes et al., 1979).
Jennische et al. (1978) found that changes in lactate
levels in moderate and extreme ischemia were not
only related to tissue acidosis but also directly
correlated with decreased membrane potentials of
ischemic tissues. Thus, there appears to be considerable evidence that lactate ion has a direct pHindependent tissue action in muscles other than
isolated canine coronary arteries.
Since lactate ion accumulation may directly participate in ischemic autoregulation in coronary vascular smooth muscle, the effects of lactate at physiological pH (7.4) and in lactic acidosis (pH 6.9-7.0)
were compared in isolated canine coronary arteries.
To test the hypothesis that lactate ion accumulation might alter Ca2+ movements and, in this manner, decrease contractile responsiveness, the inhibitory actions of lactate on tension responses to
PGF2a and KCl were ascertained, and attempts
were made to correlate these actions with specific
alterations in uptake, efflux, and mobilization of
45
Ca in the same vascular preparations. Use of KCl
772
CIRCULATION RESEARCH
as a nonspecific depolarizing agent and of P G F 2 Q as
an agent capable of mobilizing Ca2+ from binding
sites or stores in a manner similar to that of norepinephrine (NE) in other vascular smooth muscles
(for review, see Weiss, 1977a) facilitate comparison
of effects of lactate on differing Ca2+-related stimulatory mechanisms.
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Methods
Mongrel dogs of either sex (weighing 15-26 kg)
were killed by rapid intravenous injection of pentobarbital (50 mg/kg). The heart was quickly removed and placed in iced physiological saline (normal Ca2+ solution). From each heart, the proximal
and mid portions of both the left anterior descending coronary artery (LAD, 1-2 mm, o.d.) and the
circumflex coronary artery (CFX, 2-3 mm, o.d.), as
well as branches of both arteries (0.5-1.0 mm, o.d.),
were immediately removed and prepared for subsequent studies on tension (main branches only)
and Ca2+ flux. No differences in 45Ca binding and
fluxes were noted between large and small branches.
Isolated coronary vessels either were cut in a helical
fashion (Furchgott and Bhadrakom, 1953) or in
rings (Hooker et al., 1977). After dissection (no
more than 30 minutes elapsed from removal of the
heart to completion of dissection), the vascular
preparations were divided into the desired number
of sections; each muscle section was equilibrated in
normal Ca2+ solution under 1-g resting tension for
80-100 minutes prior to initiation of experimental
procedures. The normal Ca2+ solution used in these
experiments had the following composition in HIM:
NaCl, 154; KC1, 5.4; CaCl2, 1.5; glucose, 11.0; and
tris(hydroxymethyl)aminomethane, 6.0. Other solutions employed were similar except that the 1.5
mM added CaCl2 was either changed (to 0.01, 0.03,
0.1, 0.3, 5.0, or 15.0 HIM), omitted from the bathing
solution (O-Ca2+ solution), or omitted with the addition of 0.05 mM disodium ethylenediaminetetraacetate (O-Ca2+ plus 0.05 mM EDTA). All of the
preceding solutions were prepared with demineralized water, adjusted to pH 7.40 ± 0.03 with small
volumes of 6.0 N HC1, gassed with 100% O2, and
maintained at 34.0 ± 0.5°C. A different type of
solution employed in some experiments was one in
which lanthanum ion replaced isosmotically all cations present in the normal Ca2+ solution (a La3+substituted solution). The composition of this solution (Karaki and Weiss, 1979) was LaCk, 80.8 mM;
glucose, 11.0 HIM; and tris(hydroxymethyl)aminomethane, 6.0 mM. This solution was adjusted to pH
6.8 (at 0.5°C) with 1 N maleic acid (to an approximate final concentration of 3 mM). Agents studied
were potassium chloride (KC1), prostaglandin F2o
(PGF^, Upjohn), propranolol hydrochloride
(Sigma), and /-lactate (Boehringer Mannheim). Initially, it was found that approximately 1 HIM lactate
lowered the pH of a normal Ca2+ solution from 7.4
VOL. 46, No. 6, JUNE 1980
to 6.9-7.0. Thus, for all experiments, lactate was
added to the specified solution to give a concentration of 1 mM before the pH was adjusted to either
7.0 or 7.4 with small volumes of 6.0 N HC1.
Contractile responses were obtained as described
in previous studies with dog vascular smooth muscle (Hester et al., 1979; Goodman et al., 1979).
During the 80- to 100-minute equilibration period,
muscles were washed with fresh solution every 1520 minutes. Tension was measured isometrically
with a Grass model 79 polygraph and FT.03 displacement transducers. The magnitude of control
and experimental contractions was expressed in
milligrams of generated tension.
The accumulation of 45Ca (expressed as tissue:
medium ratios and net uptakes) was also measured
in this study in the manner reported previously for
vascular smooth muscle (Hudgins and Weiss, 1969;
Goodman et al., 1972). After the initial equilibration
period (90 minutes), muscle strips (under approximately 1-g resting tension) were transferred to a
specified experimental solution for 30 minutes prior
to placement in test tubes containing 10 ml of the
same solution with added 45Ca (specific activity, 0.4
^tCi/ml) for 10, 30, 60, or 90 minutes. Then, each
tissue was blotted gently with no. 5 Whatman filter
paper, dipped rapidly in four successive tubes containing 10 ml nonradioactive solution similar to the
incubation medium, blotted again, weighed on Federal-Pacific torsion balance, placed in fused quartz
crucibles, and ashed for approximately 16 hours
(overnight) in a muffle furnace at 500°C. The ash
residue was dissolved with 4 ml of 0.1 N HC1, and
aliquots from this solution were dried in appropriately prepared planchets and then counted in Nuclear-Chicago proportional gas-flow counters with
ultrathin windows. The 45Ca tissue:medium (T/M)
ratio (ml/g) was obtained by dividing the amount
of 45Ca taken up per gram wet weight of tissue by
the concentration of 45Ca per milliliter of incubation
solution. The values for 45Ca T/M ratio can be
converted to calculated total Ca2+ uptake units
(micromoles Ca2+ per gram muscle wet weight) by
multiplying the ratio values by the total concentration of extracellular Ca2+ (micromoles per milliliter). The total Ca2+ concentration was calculated
from the amount of CaCl2 added to the bathing
solution and by estimating the total amounts of
trace Ca2+ present in other solution constituents
and the amount of 45Ca (plus accompanying carrier
Ca2+) that was added to the solution. The concentration was a maximum of 0.001 mM in Ca-free
solutions and this value plus that of the added Ca2+
in varied Ca2+ solutions.
In experiments measuring [14C]lactate or [14C]sucrose T/M ratios (specific activity, 0.4 juCi/ml),
vascular strips were equilibrated as described above
and then placed into tubes containing normal Ca2+
solution plus [14C]lactate at either 0°C or 34°C for
pH-INDEPENDENT EFFECTS OF LACTATE IN CORONARIES/ifester et al.
time) or as rate coefficient curves (which express
the decline of tissue 45Ca content as a percentage of
the 45Ca present in the tissue during each time
interval). Student's t-tests for paired and unpaired
data were used. A probability of less than 0.05 was
considered significant.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
specified intervals. Then, tissues were removed
from the radioactive solution, blotted, dipped, and
weighed in a manner similar to that employed for
45
Ca T/M ratio determinations. The muscles were
wet-ashed in 2 ml of 1 N NaOH at 80°C (90-180
minutes), and the solution was neutralized with
appropriate amounts of 2 N HC1 and diluted to 5 ml
with demineralized water. Aliquots from these solutions were placed into planchets, evaporated to
dryness, and counted as described previously. The
[14C]lactate T/M ratio was calculated in the same
manner as the 45Ca T/M ratio.
The La3+-resistant 45Ca uptake was measured as
described previously (Karaki and Weiss, 1979) after
60-minute washout of 45Ca into an isosmotic La3+substituted (80.8 mM LaCla) solution. After 45Ca
incubation procedures similar to those in 45Ca T/M
ratio experiments (including specified portions of
45
Ca incubation periods with lactate, PGF2o, and
KCl), muscle strips were placed into test tubes
containing 15 ml isosmotic La3+-substituted solution at 0.5°C for 60 minutes (sequential periods of
30 seconds, 9.5 minutes, 10 minutes, 20 minutes,
and 20 minutes). They then were blotted, weighed,
and ashed as in 45Ca uptake experiments.
Muscle strips employed in 45Ca washout experiments were tied with monofilament 000 nylon
thread and attached under approximately 1 g of
tension to stainless steel rods in the same manner
as had been employed in the uptake experiments.
The muscles, after equilibration for 60 minutes in
either O-Ca2+ or normal Ca2+ solution containing
45
Ca, were blotted and dipped as in 45Ca T/M experiments and then transferred to tubes containing
2 ml of the aerated, nonradioactive solution. The
bathing solution was collected in planchets and
replaced at specified intervals for the duration of
the washout. Each 2-ml sample was evaporated to
dryness before being counted. On completion of the
washout, each muscle was blotted, weighed, ashed,
and prepared for counting as in 45Ca uptake experiments. The data obtained were expressed (Bianchi,
1965; Weiss, 1966) as either desaturation curves
(which illustrate the decline of tissue 45Ca with
Results
As shown in Table 1, prior treatment (15 or 30
minutes) of canine coronary arteries in a normal
Ca2+ solution with 1 mM lactate (normal pH, 7.4)
depresses subsequent contractile responses to 10
JUM PGF2a more than those to 60 mM KCl. This
table also shows that lactate at decreased pH (6.97.0, lactic acidosis) causes no greater depression of
KCl-induced contractile responses than lactate at
pH 7.4. Contractile responses induced by PGF2a,
KCl, or 0.1 mM NE (after 20-minute exposure to 1
JUM propranolol) are relaxed by the subsequent addition of lactate (accompanied by a decrease in pH,
6.6-6.9) to the bathing solution. The responses to
PGF2« (1.11 ± 0.06 to 0.52 ± 0.05 g, 53.2% relaxation)
and NE (0.85 ± 0.10 to 0.51 ± 0.11 g, 40.0% relaxation) are decreased more than are responses to KCl
(2.0 ± 0.14 to 1.83 ± 0.15 g, 8.5% relaxation).
Responses of canine coronary arteries to PGF2Q
are more resistant to the effects of Ca2+ depletion
than are responses to KCl (Table 2). After only 5
minutes in O-Ca2+ solution, KCl-induced responses
are depressed by 59%, whereas responses to PGF2a
are depressed only by 21%. After 30-minute incubation in O-Ca2+ solution, KCl-induced responses
are depressed by 81%, and responses induced by
PGF2a are depressed by 62% (not significantly different after 30 minutes).
Differences in Ca2+ uptake into canine coronary
arteries from O-Ca2+ or normal Ca2+ solutions are
illustrated in Table 3. These values are expressed
as both T/M ratios (ml/g) or as net Ca2+ uptake
(/unoles/g) which includes a correction for the 14Cextracellular space (0.339 ml/g, Goodman et al.,
1979). Equilibration occurs more rapidly in normal
Ca2+ solution (almost complete in 10 minutes) than
in O-Ca2+ solution (30-60 minutes). The slower
TABLE 1 Effect of Pretreatment of Canine Coronary Arteries with 1.0 mM Lactate on
Responses to Prostaglandin F^ or Potassium
Prior
Agonist*
PGF^
PGF^
KCl
KCl
KCl
KCl
Contractile response (mg ± SE)t
with lactate
(min)
Solution
15
30
15
30
15
30
7.4
7.4
7.4
7.4
6.9-7.0
6.9-7.0
pH
Control
(1.5 HIM Ca2*)
1087.5
1250.0
1441.7
1293.5
1245.0
1492.2
773
±
±
±
±
±
±
92.9
162.8
131.9
233.9
120.5
172.9
In 1.0 mM
lactate
625.0
600.0
1220.0
1035.0
970.0
1146.9
±
±
±
±
±
±
87.6
178.9
131.8
224.6
100.6
135.0
contractile response
(% ± SE)
40.6
40.4
16.4
20.8
23.0
22.4
±
±
±
±
±
±
7.8t
7.6*
4.0
3.8
2.8§
2.5§
* Concentrations employed were 10 JIM prostaglandin F&, (PGF&,) and 60 mM potassium (KCl), n = 8-30.
f In all cases, 1.0 mM lactate significantly decreased the contractile response (paired <-test, P < 0.005).
i Significantly different from corresponding values for KCl at pH 7.4 (unpaired t-test, P < 0.02).
§ Not significantly different from values for KCl at pH 7.4.
CIRCULATION RESEARCH
774
VOL. 46, No. 6, JUNE 1980
2+
TABLE 2 Effects of Ca Depletion on Contractile Responses to 10 \IM PGF^ and 60
mM KCl in Canine Coronary Arteries*
Contractile response (mg ± SE)
2+
Agonist
0-Ca
(min)
5
5
15
15
30
30
PGFj.
KCl
PGF^
KCl
PGF2.
KCl
2+
Control (1.5 mM Ca )
937.5 ±
840.0 ±
1035.0 ±
1005.8 ±
941.6 ±
1106.3 ±
99.4
186.0
210.7
87.1
189.0
160.8
Decrease in
2+
In 0-Ca
<% ± SE)
731.3+ 93.5
370.0 ± 151.0
595.0 ±111.2
218.4 ± 36.1
366.6 ± 95.5
218.8 ± 92.1
21.0 ±
58.8 ±
41.3 ±
76.1 ±
61.7 ±
80.8 ±
7.1
6.3
6.3
3.5
7.1
6.9
• n = 6-8.
f Unpaired values for PGF^ and KCl are significantly different (P < 0.005) at only 5- and 15-minute incubation
intervals.
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loading rate in a O-Ca2+ solution may be associated
with equilibration at slower loading high affinity
Ca2+-binding sites as has been described for rabbit
aorta (Weiss, 1977b, 1978).
The effect of lactate (pH 7.4) on the loss of 45Ca
from previously loaded canine coronary arteries is
shown in Figure 1. If the washout was into either a
O-Ca2+ or O-Ca2+ plus EDTA (0.05 mM) solution,
the rate of loss of 45Ca was decreased by addition of
lactate. In a O-Ca2+ solution, the addition of lactate
resulted in a 35% increase in the graphically calculated half-time (ti/2) value (from 36.0 to 48.5 minutes). In a O-Ca2+ plus EDTA solution, which permits a closer approximation of net efflux of 45Ca by
minimizing the influence of 45Ca reuptake (Goodman et al., 1972), the addition of lactate results in
a 51% increase in the graphically calculated ti /2 from
14.6 to 22.0 minutes). This persistence of the lactate-induced effect in EDTA-containing solutions
indicates that lactate alters net efflux of 45Ca rather
than reuptake mechanisms. Also, the addition of
lactate accompanied by a decreased pH during 45Ca
washout into a O-Ca2+ solution (figure not shown)
results in a similar (37%) increase in the graphically
calculated ti / 2 (from 31.0 to 42.5 minutes, n = 8) as
at pH 7.4. Although control ty 2 rates of 45Ca washout may vary among tissues, and averaged rates are
not identical, lactate addition during 45Ca washout
TABLE 3 Equilibration ofibCa in Canine Coronary
Arteries in Solutions Containing 1.5 mM Ca?* or No
Added Co2*
Ca 2+
(mM)*
"Ca incubation
duration (min)
n
"Ca T/M ratio
(ml/g ± SE)
0.001
0.001
0.001
0.001
1.501
1.501
1.501
1.501
10
30
60
90
10
30
60
90
18
12
12
10
8
9
20
7
9.866 ±
15.341 ±
19.395 ±
19.658 ±
1.315 ±
1.330 ±
1.366 ±
1.299 ±
0.516
1.450
0.842
1.971
0.046
0.057
0.028
0.057
Ca2+ uptake
(/unol/g)t
0.0095
0.0150
0.0191
0.0193
1.463
1.487
1.541
1.440
• Either no added Ca2* (O-Ca2* solution with 0.001 mM Ca2+) or normal
solution (1.501 mM Ca 2+ ).
•f Calculated from *5Ca T / M ratios by first subtracting the extracellular
space (["C]-sucrose T / M ratio, 0.339 ml/g) and then multiplying the
remainder by the extracellular Ca2* concentration.
always increased slow component ti/2, whereas control 45Ca washout did not change.
The effect of temperature on the uptake of
[14C]lactate into canine coronary arteries is shown
in Figure 2. Uptake at 34°C continues to increase
for at least 120 minutes. Lactate appears to enter
the cell rapidly; it then is metabolized, and the Relabeled metabolites accumulate. When the temperature is decreased to 0°C (inhibiting metabolismdependent transport mechanisms), equilibration occurs rapidly to a value not much greater than the
extracellular T/M ratio, 0.339 ml/g.
The addition of Ca2+ (0.03 mM) to a washout of
45
Ca into a O-Ca2+ solution results in a rapid and
maintained increase in the rate of loss of 45Ca (Fig.
100
80
60
UJ
^100
2
80
Without Lactate
With Lactate, 1.0mM
40
20
5
10
15
20
25
MINUTES
FIGURE 1 Effect of addition of lactate (1 mM) (pH 7.4)
after 15 minutes of ibCa washout on the efflux of i5Ca
from canine coronary arteries into a O-Ca2+ solution
(upper panel) or into a O-Ca2+ plus EDTA solution
(lower panel). Each preparation was incubated for 60
minutes in a O-Ca2+ solution plus 4bCa before initiation
of the washout. Each desaturation curve represents the
average values for either 12 (upper panel) or nine (lower
panel) muscle strips. Broken lines indicate extrapolation
of the slow component of the washout.
775
pH-INDEPENDENT EFFECTS OF LACTATE IN CORONARIES /Hester et al.
1.4r
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Canine Coronary Artery
60
MINUTES
90
120
FIGURE 2 Effect of temperature on the uptake of
[>4C]lactate into canine coronary arteries over time.
Points on each curve represent the mean ± SE of eight to
11 muscle strips.
3). This procedure gives an estimate of exchangeability between Ca2+ and 45Ca. In the presence of
lactate (pH 7.4), the addition of Ca2+ (0.03 HIM)
results in an increased rate of 45Ca loss that is
similar to but lower than that in the absence of
lactate.
Values for 45Ca T/M ratios and calculated Ca2+
X
2
5
UJ
8
in
uptakes in canine coronary arteries at different
extracellular Ca2+ concentrations are summarized
in Table 4. By use of Scatchard plots (Scatchard,
1949) of Ca2+ uptake in a manner similar to that
first used for rabbit aorta by .Weiss (1977b), the
uptake of Ca2+ by canine coronary arteries can be
separated into two distinct binding sites, a high and
a low affinity site (Fig. 4). The x-axis intercept
yields an estimate of the number of high or low
affinity Ca2+-binding sites, while the affinities of the
two sites can be expressed in terms of the apparent
dissociation constants, KD (X intercept/y intercept).
Rabbit aortic smooth muscle (Weiss, 1978) has approximately twice as many high affinity Ca2+-binding sites as does canine coronary artery (1.58 vs.
0.78), whereas the KD values, as well as the number
of low affinity sites (9.00 vs. 8.53), are approximately
equal. The addition of lactate (pH 7.4) to a solution
maximizing high affinity Ca2+ binding (0.03 HIM
Ca2+; the middle point on the high affinity component in Fig. 4) increases the rate of 45Ca uptake
(nonequilibrated 10-minute incubation values)
without altering the maximum uptake (30-minute
equilibrated values) at high affinity Ca2+-binding
sites (Table 5). On the other hand, as the concentration of Ca2+ in the 45Ca incubation solution is
increased 10-fold (to 0.3 HIM) and the relative
amount of low affinity Ca2+ binding is increased,
the effect of lactate on the rate of 45Ca uptake is no
longer observed.
To further define the effects of lactate at pH 7.4
on binding of Ca2+ in canine coronary arteries, La3+resistant 45Ca fractions were measured. An isosmotic La3+-substituted solution at 0.5 °C was used
to define these residual Ca2+ fractions as has been
done in other vascular smooth muscles (Karaki and
Weiss, 1979; H Karaki, RK Hester, GB Weiss, unpublished observation). Solutions with much lower
concentrations of La3+ readily block Ca2+ influx and
remove loosely bound Ca2+ from a variety of tissues
TABLE 4 Tissue:Medium Ratios and Uptake Values
for 45Ca at Different Extracellular Ca2* Concentrations
in Canine Coronary Arteries*
li. . _
O E
Without Lactate
LL.
LJ.
LLJ
O
O
With Lactate, I.OmM
-0-Ca++-
UJ
Si
• 4 — 0.03 mM Co + + • '
5
10
• '
15 20
15 20
MINUTES
FIGURE 3 The effect of 1 mM lactate (pH 7.4) on the
increase in rate coefficient of4bCa caused by addition of
0.03 mM Ca2* during washout of canine coronary arteries
into a O-Ca2* solution. Each preparation was incubated
for 60 minutes in a O-Ca2+ solution plus *bCa before
initiation of the washout. The two plotted rate coefficient
curves represent the averaged values from five paired
experimental 4bCa washouts.
45
Ca T/M
ratios
(ml/g ± SE)
Extracellular
Ca2+ (mM)t
0.001
0.011
0.031
0.101
0.301
1.501
5.001
15.001
39
22
10
29
26
35
11
12
19.395
12.646
10.860
5.082
2.720
1.717
1.178
0.775
±
±
±
±
±
±
±
±
0.842
0.707
0.660
0.183
0.127
0.052
0.090
0.028
Calculated
Ca2* uptake
Oimol/g):):
0.001
0.135
0.326
0.479
0.717
2.068
4.195
6.540
' Incubated with <5Ca-containing solution for 60 minutes before analysis.
t Includes 0.001 mM Ca2+ present in *sCa solution containing no added
Ca2+.
% Calculated as the product of the extracellular Ca2* concentration
and the "Ca T / M ratio minus the extracellular space (["C]-sucrose T / M
ratio, 0.339 ml/g).
CIRCULATION RESEARCH
776
20
r
VOL. 46, No. 6, JUNE 1980
Canine Coronary Artery
Site
High Affinity
Low Affinity
"5
X-axis Intercept Calculated KQ Value
0.78
0.041
4.847
8.53
+
o
o
UJ
Ul
12
or
u.
• 80.8 mM Lo+++ solution-
8
LL. L L -^
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U. LJ E
U.LJ
E
o
o
Canine Coronary Artery
40
60
80
100
120
5 Effect of La3*-substituted (80.8 IUM) solution
at 0.5° C on 45Ca efflux from canine coronary arteries.
Desaturation (upperpanel) and corresponding rate coefficient (lower panel) curves were obtained from averaged
washouts of the same 12 muscles. The broken line indicates extrapolation of the slow component of the desaturation curve (upper panel). All muscles were incubated
in normal Ca2+ solution plus 45Ca for 60 minutes prior to
the washout.
FIGURE
2
4
6
8
10
BOUND Ca ++ (^mol/g)
*5
FIGURE 4 Scatchard plot of Ca uptake into canine
coronary arteries after 60-minute incubation in solutions
containing different Ca2+ concentrations. Each point
represents the average of 12-30 muscle strips.
(see Weiss, 1974). Figure 5 demonstrates that La3+substituted solution at 0.5°C almost completely inhibits Ca2+ efflux in canine coronary arteries within
60 minutes. The calculated ti /2 for the slow component of the 45Ca desaturation curve under these
conditions is greater than 462 minutes, and the rate
coefficient decreases to approximately 0.15%/min.
Similar results have been reported for rabbit aorta
(Karaki and Weiss, 1979) and canine renal arteries
(H Karaki, RK Hester, GB Weiss, unpublished
observation) and veins (RK Hester and GB Weiss,
unpublished observation). Comparison of values
5 Effect of 1 MM Lactate on A5Ca Uptake from
0.03 mM and 0.3 mM Ca2+ Solutions in Canine
Coronary Arteries*
TABLE
"Ca T/M ratios (ml/g ± SE)
45
(mM)
Ca incubation
duration (min)
0.03
0.03
0.3
0.3
10
30
10
30
Control
5.768 ±
8.329 ±
2.686 ±
3.184 ±
0.346
0.599$
0.231
0.238
Lactate
7.172
8.510
2.668
2.809
±
±
±
±
0.439t
0.442
0.220
0.125
' n = 18 (0.03 mM) or 8 (0.3 mM).
t Significantly (P < 0.025) different from paired control without lactate.
% Significantly (P < 0.005) different from paired control at 10 minutes.
from Tables 4 and 6 demonstrates that exposure to
La3+-substituted solution at 0.5°C for 60 minutes
decreases the relative lo.w affinity Ca2+-binding sites
(in 5.0 mM Ca2+) in canine coronary arteries by
95.2% (1.178 ± 0.090 to 0.057 ± 0.009 ml/g), while
decreasing the high affinity Ca2+-binding sites (in
0.03 MM Ca2+) by 91.6% (10.860 ± 0.660 to 0.917 ±
0.071 ml/g).
The 45Ca remaining after a 60-minute washout in
La3+-substituted solution at 0.5°C (expressed as the
residual Ca2+ uptake or the La3+-resistant 45Ca tissue: medium ratio) is summarized in Table 6 for
Ca2+ under both high affinity (0.03 mM extracellular
Ca2+) and low affinity (5.0 mM extracellular Ca2+)
conditions. High affinity residual Ca2+ uptake is
significantly decreased by PGF2a (28.8%), significantly increased by lactate (25.8%), and unaffected
by KC1. The lactate-induced increase in high affinity residual Ca2+ uptake did not occur when KC1
was also present. Low affinity residual Ca2+ uptake
was significantly increased by KC1 (70.2%) and was
not affected by PGF2<, or lactate. However, lactate
did block the KCl-induced increase in low affinity
residual Ca2+ uptake. Thus, PGF2« and lactate had
opposite effects on high affinity Ca2+ and no effect
pH-INDEPENDENT EFFECTS OF LACTATE IN CORONARIES/i/ester et al.
777
6 Effects ofPGF^ KCl, and Lactate on High and Low Affinity Residual Ca2*
Uptake in Canine' Coronary Arteries
TABLE
4
°Ca incubation conditions*
La3+-resistant <5Ca T/M
ratio (ml/g ± SE)
Change in
residual Ca2* (%)
Pi
High affinity (0.03 mM Ca *):
0.917 ± 0.071
11
7
0.653 ± 0.060
5
1.012 ± 0.051
10
1.154 ± 0.052
5
0.897 ± 0.039
-28.8
+10.4
+25.8
-2.2
<0.001
>0.2
<0.02
>0.8
Low affinity (5.0 mM Ca2*):
11
0.057 ± 0.009
9
0.063 ± 0.010
5
0.097 + 0.008
11
0.060 ± 0.007
5
0.059 ± 0.007
+10.5
+70.2
+5.3
+3.5
>0.6
<0.01
>0.7
>0.8
n
2
Control
+ PGF2a
+ KC1
+ lactate
+ KCl + lactate
Control
+ PGF 2 D
+ KC1
+ lactate
+ KCl + lactate
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* All muscles were incubated with 45Ca for 60 minutes. Lactate was present for the full 60-minute <5Ca incubation
period; KCl and PGF^ were added for only the final 10 minutes of the 60-minute incubation. Concentrations of
agents employed are PGF^, 10 JIM; KCl, 60 mM; lactate, 1.0 mM at pH 7.4.
f The level of significance (P) is calculated for grouped nonpaired values.
on low affinity Ca2+, whereas KCl increased low
affinity Ca2+ but did not significantly alter high
affinity Ca2+. Lactate and KCl together appeared to
prevent the effects on residual Ca2+ obtained in the
presence of only one of these agents.
Discussion
The inhibitory effects of lactate on contractility
and 45Ca mobilization in isolated canine coronary
arteries are independent of concomitant changes in
pH (acidosis). Lactic acidosis caused a slightly more
rapid depression of KCl-induced responses in canine coronary arteries than did lactate at pH 7.4,
but the extent of inhibition of contraction was essentially equal. Turnheim et al. (1977) found that
acidosis appeared to depress the contractile process
of canine coronary arteries directly. The decreases
in 45Ca loss from canine coronary arteries observed
with both lactic acid-induced acidosis and lactate
ion accumulation at pH 7.4 were quantitatively
similar. Thus, lactate ion appears to have a direct
pH-independent action in isolated canine coronary
arteries.
The KCl-induced contractile responses in canine
coronary arteries were sensitive to the effects of
Ca2+ depletion. This finding is similar to results for
many other vascular prejtarations in that KCl-induced responses are primarily dependent on an
influx of Ca2+ for development of tension (for reviews, see Weiss, 1975, 1977a). Responses to PGF2a
in isolated canine coronary arteries are much less
sensitive to the effects of a Ca2+-deficient solution,
especially for durations of exposure of less than 30
minutes. Apparently, PGF2a is capable of mobilizing
Ca2+ from less readily depleted sites or stores and,
in this manner, initiating tension responses in canine coronary arteries. This capability resembles
that noted for NE in other vascular smooth muscles
(for review, see Weiss, 1977a). Thus, characteriza-
tion of the effects of an inhibitor such as lactate on
responses to KCl and PGF2Q should provide as
useful a comparison in isolated canine coronary
arteries as does the comparison of effects of NE and
KCl in other vascular tissues. In this study, prior
treatment with lactate depressed a subsequent contraction induced by PGF2a more than one elicited
with KCl. Furthermore, the subsequent addition of
lactate to muscles previously contracted with
PGF2o or NE (in the presence of propranolol) are
relaxed to a greater degree than are contractions
obtained with KCl. In this latter situation, the
direct effects of a decrease in pH (which accompanied the addition of lactate) cannot be excluded.
However, these findings to indicate that lactate has
an enhanced effect on responses to agonists which
mobilize less readily depleted cellular stores of Ca2+.
The kinetics of [14C]lactate uptake indicate that
this organic anion is rapidly taken up into isolated
canine coronary arteries. The uptake appears to be
an active process, since the major portion of cellular
uptake is inhibited by decreasing the temperature
to 0°C. Lactate transport into erythrocytes has
been shown to occur primarily via a specific monocarboxylate transport system (distinct from an organic anion exchange channel) probably by an H + lactate transport (symport) system which operates
as a function of H + gradients (Dubinsky and
Racker, 1978). Since the extracellular equilibration
and cellular uptake of lactate is rapid, its actions
could occur at or within the cell membrane or even
intracellularly.
To evaluate the specific effects of lactate on Ca2+
distribution and mobilization in isolated canine coronary arteries, it was first necessary to establish
certain baseline parameters for this tissue. In a
previous study (Goodman et al., 1979), 45Ca efflux
from canine coronary arteries was quantified and
analyzed in a manner similar to that employed for
778
CIRCULATION RESEARCH
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other smooth muscles (essentially separated into a
two-component desaturation curve). Lactate ion
and lactic acidosis in canine coronary arteries both
result in a decrease in loss of slow component 45Ca
which is quantitatively similar to that obtained with
hypertonic mannitol in the same preparations
(Goodman et al., 1979). Poole-Wilson and Langer
(1979) have shown that acidosis not only depresses
developed tension in isolated perfused rabbit interventricular septum but also inhibits efflux of slowly
exchanging 45Ca and uptake of 47Ca. Since the lactate-induced decrease was observed in both O-Ca2+
and O-Ca2+ plus EDTA solutions, it probably represents an effect on net efflux rather than on rebinding or reuptake of Ca2+. A solution containing
O-Ca2+ plus a trace of EDTA would decrease reuptake by chelating the emerging 45Ca and preventing
back flux or reuptake (Goodman et al., 1972). Furthermore, the presence of lactate ion inhibits the
increase in exchangeability of 45Ca in isolated canine
coronary arteries following addition of nonradioactive Ca2+ during 45Ca efflux into O-Ca2+ solutions.
Lactate may decrease Ca2+ efflux and exchangeability by increasing binding of Ca2+ at PGF2a-sensitive sites.
The uptake of 45Ca by isolated canine coronary
arteries is relatively rapid in normal Ca2+ solutions
with equilibration being attained within approximately 10 minutes. Conversely, in a O-Ca2+ solution,
the rate of uptake is slower (equilibration within
approximately 60 minutes). By varying the concentration of added extracellular Ca2+ from 0 to 15.0
HIM, we have shown through subsequent analysis of
Scatchard plots that the canine coronary arteries
have two distinctly separate Ca2+-binding sites, exhibiting relatively high or low binding affinities. In
this respect, the canine coronary arteries are qualitatively similar to rabbit aorta (Karaki and Weiss,
1979) and canine renal artery and vein (RK Hester
and GB Weiss, unpublished observation). At lower
extracellular Ca2+ concentrations, the binding of
Ca2+ occurs primarily at high affinity sites, whereas
at higher extracellular Ca2+ concentrations, binding
at the quantitatively larger low affinity sites is
optimized. The addition of lactate to a solution
which maximized high affinity Ca2+ binding (0.03
mM Ca2+) results specifically in an increase in the
rate of saturation of high affinity Ca2+-binding sites
without altering the maximum number of sites
available. Since the rate of binding to high affinity
sites was increased and not the apparent maximum
number of sites, an increased affinity of Ca2+ for
these sites may be associated with the presence of
lactate. As the extracellular Ca2+ concentration is
increased (concomitantly increasing the predominant influence of the quantitatively larger low affinity Ca2+ binding on the measurable 45Ca uptake),
the effect of lactate on high affinity Ca2+ binding is
obscured.
VOL. 46, No. 6, JUNE 1980
In rabbit aortic smooth muscle (Karaki and
Weiss, 1979), a similar La3+-substituted solution not
only inhibits Ca2+ influx and superficial Ca2+ binding but also almost completely inhibits Ca2+ efflux.
If vascular muscles are washed out in this solution,
a reasonable measurement of residual cellular Ca2+
(La3+-resistant 45Ca uptake) can be obtained. In this
manner, quantitatively small changes in residual
Ca2+ elicited with agonists or antagonists before
washout in La3+-substituted solution at 0.5°C can
now be ascertained. It is possible to attempt to
correlate these changes with alterations in contractile tension observed under similar conditions.
PGF2a decreases the high affinity residual Ca2+; this
can be related to a shift in La3+-resistant Ca2+ from
high affinity sites partly to other more superficial
sites (preceding removal by La3+). Simultaneously
(and undetected by 45Ca techniques employed), part
of the high affinity La3+-resistant Ca2+ is shifted to
the myoplasm (resulting in increased intracellular
Ca2+ and contractile protein activation). This latter
effect could correlate with the remaining PGF2ainduced tension response obtained in Ca2+-deficient
solutions. This type of correlation reemphasizes the
similarity of the action of PGF2a to that of NE in
rabbit aorta (Karaki and Weiss, 1979). In rabbit
aortic smooth muscle, NE releases high affinity
residual Ca2+ and, in this manner, mobilizes a La3+resistant pool of Ca2+ to increase contractility. Conversely, KC1 acts primarily to increase low affinity
residual Ca2+ in isolated canine coronary arteries.
In rabbit aorta (Karaki and Weiss, 1979), KC1 also
primarily causes a large increase in low affinity
residual Ca2+. Even though the action of KC1 on
45
Ca retention in canine coronary artery is similar
to that in rabbit aorta, canine coronary arteries
have a more labile (less La3+-resistant) low affinity
Ca2+ fraction.
Thus, lactate increases the binding of high affinity residual Ca2+, and this increase is approximately
equal in magnitude to the decrease caused by
P G F 2 Q . Since lactate has a greater effect on tension
responses to PGF20 than to KC1, this increase in
residual Ca2+ at high affinity sites may effectively
inhibit the PGF2a-induced release (or decrease) of
Ca2+ from this same fraction. This cellular store of
Ca2+ may include that Ca2+ mobilized by PGF2 in
canine coronary arteries to initiate tension. The
relationships between effects of lactate and those of
KC1 on residual Ca2+ may be less direct. Lactate
has no effect on low affinity residual Ca2+ levels but
inhibits the KCl-induced increase in low affinity
residual Ca2+ . Since lactate has minimal effects on
KCl-induced tension responses in isolated canine
coronary arteries, this KCl-induced increase in low
affinity residual Ca2+ may be directly related to
changes in polarization, and lactate may only indirectly affect subsequent binding and removal of this
Ca2+. Depolarization (added KC1) also prevents the
pH-INDEPENDENT EFFECTS OF LACTATE IN CORONARIES/Hester et al.
lactate-induced increase in residual high affinity
Ca2+, possibly by decreasing affinity of these sites
for Ca2+. However, in polarized muscle, lactate acts
in a pH-independent manner to decrease contractile
responsiveness by inhibition of mobilization of high
affinity Ca2+.
Acknowledgments
The technical assistance of Nancy S. Mahanay is gratefully
acknowledged. Prostaglandin F2a was generously supplied by Dr.
John Pike of the Upjohn Company, Kalamazoo, Michigan.
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Circ Res. 1980;46:771-779
doi: 10.1161/01.RES.46.6.771
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