314
Force, Membrane Potential, and [Ca2j]i During
Activation of Rat Mesenteric Small Arteries
With Norepinephrine, Potassium, Aluminum
Fluoride, and Phorbol Ester
Effects of Changes in pH;
Peter E. Jensen, Alun Hughes, Harrie C.M. Boonen, Christian Aalkjaxr
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In activated rat mesenteric small arteries, the effect of pH1 on force, membrane potential, and free
cytosolic calcium ([Ca2"]i) was assessed. Arteries were mounted in a myograph for isometric force
development, and [Ca2"]j, pH;, or membrane potential was measured simultaneously with force. During
activation with norepinephrine, potassium, aluminum fluoride (AIF,), and phorbol 12-myristate 13acetate (PMA, a phorbol ester), the vessels depolarized and [Ca2+], increased, although the ratio of force
to [Ca2+]j was less during potassium activation than with the other types of activation. Changes in pH1,
with a constant pH. were induced with NH4Cl or by changing Pco2. In resting vessels, the effects of the
changes in pH; on tension, membrane potential, and [Ca21]i were negligible. In vessels activated with
norepinephrine or AMF4, alkalinization caused an acute decrease of tone, which could be explained by a
decrease in [Ca 2+1i consequent to repolarization of the membrane. In vessels activated with potassium or
PMA, the effects of alkalinization were smaller. This is consistent with acute alkalinization, affecting steps
proximal in the excitation-contraction coupling distal to activation of G proteins. Acidification caused a
transient increase in tone and [Ca 2+11, irrespective of the mode of stimulation, without affecting the
membrane potential. Ryanodine did not abolish the transient increase in tone and [Ca 2+];. Thus, acute
intracellular acidification may induce tone by release of an intracellular ryanodine-insensitive calcium
pool or by affecting transmembranal calcium flux although in a membrane potential-independent way.
(Circulation Research 1993;73:314-324)
KEY WORDs * pH1 * free cytosolic calcium * membrane potential * mesenteric arteries
T he main vascular effects of reduction and increase
in pH are vasodilation and vasoconstriction,
respectively. However, it was reported almost 30
years ago that acute acidification and alkalinization give
a paradoxical initial transient vasoconstriction and vasodilation, respectively, in vivo.12 Similar acute effects of
changes in pH have more recently been reported in in
vitro experiments,3-8 in which acute intracellular acidification and alkalinization have been seen to induce
transient contraction and relaxation, respectively, in different isolated vessels. The intracellular acidification and
alkalinization were induced by either changes in Pco2 or
incubation with NH4C1. However, although the constriction induced by acidification was seen in vessels activated
with either agonists or potassium, the vasodilation seen
with alkalinization was mainly seen in vessels activated
Received December 7, 1992; accepted April 5, 1993.
From the Department of Pharmacology and Danish Biomembrane Research Centre, Bartholin Building, University of Aarhus
(Denmark) (P.E.J., C.A.); the Department of Clinical Pharmacology, St. Mary's Hospital, London, UK (A.H.); and the Department of Pharmacology, University of Limburg, Maastricht, The
Netherlands (H.C.M.B.).
Reprint requests to Department of Pharmacology and Danish
Biomembrane Research Centre, University of Aarhus, DK-8000
Aarhus C, Denmark (Dr Aalkjer).
with agonists.367 The mechanisms responsible for these
effects of acute changes in pHi remain unknown. The
purpose of this study was twofold: (1) to compare the
effect of norepinephrine, potassium, aluminum fluoride
(AIF4), and phorbol 12-myristate 13-acetate (PMA) on
force, membrane potential, and [Ca2+]i in rat mesenteric
small arteries and (2) to investigate the mechanisms
responsible for the paradoxical acute effects of changes
in pH1 on vessel tone. This was done by assessing the
effects of changes in pH, measured with 2',7'-bis(2carboxyethyl)- 5(6)-carboxyfluorescein (BCECF) on
membrane potential (measured with microelectrodes),
[Ca2+]i (measured with fura 2), and isometric force in
vessels activated with norepinephrine, potassium, AIF4,
and PMA. The latter two modes of activation were used
to bypass the agonist-receptor-coupling step. Changes in
pH, were induced by NH4Cl or by changes in Pco2 while
pH, was maintained constant.
Materials and Methods
Tissue
Arteries with an internal diameter of approximately
200 ,um were dissected out from the mesenteric bed of
C02-killed 12- to 14-week-old male Wistar rats and
used in all experiments.
Jensen et al Effects of Changes in pH; on Tone
A
6
3 pM norepinephrine
10 % CO2
10%C02,g 0%C02-
315
2.5 pM norepinephrine
10 mM NH4C1
B
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~l
l
5
54
0
3
0
2
0
0
0
0
it
2
1
0
15 min
15 min
7.60
7.35
7.35
7.10
7.10[
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6.85F
6.85 L
6.60L
FIG 1. Recordings showing effects of changes in Pco2 (panel A) and NH4Cl (panel B)
on
pHi in vessels activated with
norepinephrine.
Measurements of Force and [Ca2+i and
of Force and pHi
Simultaneous measurements of [Ca'+]i and force and
of pH, and force were obtained as described previously.910 In brief, vessel segments (approximately 2 mm
long) were mounted in a myograph for isometric force
measurements.1" The internal circumference of the
mounted vessel was normalized on the basis of the
passive-tension length curve to a value that was 0.9
times the circumference the vessel would have had in
vivo under a transmural pressure of 100 mm Hg. At this
setting, near-maximal force development can be obtained.1' The vessels never exhibited myogenic tone,
and they were fully relaxed when incubated in physiological salt solution (PSS; for composition, see below).
The myograph was then placed on a microscope, and
the vessels were loaded for 4 hours at room temperature
with 10 ,uM fura 2-AM or for 1 hour at 37°C with 5 ,uM
of the acetoxymethyl ester of BCECF (BCECF-AM) for
measurements of [Ca 2]i and pHi, respectively. For
[Ca2+]i, fura 2-AM was washed out,
the myograph was heated to 37°C and placed on a Zeiss
inverted microscope, and the vessel was excited with
light from a 75 -W xenon lamp that passed through a
347-nm band-pass filter or a 380-nm band-pass filter
(bandwidth, 5 nm). The emission from the preparation
was collected by epifluorescence through a 500- to
530-nm band-pass filter and a <720-nm cutoff filter.
The emission signals when excited with 347- and
380-nm light were stored, together with force measurements, on computer. After completion of the experimental protocol, the signals were calibrated as previously described9 using the intracellular dissociation
constant (Kd) of 342 nM for the fura 2-Ca2 complex.12
The Kd for the fura 2-Ca`+ complex may be affected by
pH.'3-15 Alkaline pH may cause no change or a small
decrease of Kd, whereas Kd increases with decreasing
pH.13-'5 We did not correct for these effects, partly
because we only induced relatively small changes in pH,
and partly because these effects on Kd would only result
in an underestimation of the [Ca `]i transients we report
but no qualitative errors. In experiments in which
[Ca'+], was measured, the solution was changed by
vacuum suction, followed by addition of new solution.
For measurement of pHi, the myograph was placed
on a Zeiss photomicroscope and excited with light from
a 75 -W xenon lamp that led into a Zeiss monochromator to provide 450- and 495 -nm light. The emission from
the preparation was collected by transillumination
through a x40 water immersion objective, a 540-nm
band-pass filter, and a <720-nm cutoff filter. The ratio
of emission when excited with 495-nm and 450-nm light
was calculated after subtraction of the background
fluorescence and, together with force measurements,
was stored on computer. The ratio was calibrated with
nigericin as described previously.1016 The solution was
measurements of
10
mM
NH4CI
-
-E
z
0
c
+.
200
X
125k
50
1S min
FIG 2. Recordings showing effects of NH4CI
tension in a relaxed vessel.
on
[Ca 2+]i and
316
Circulation Research Vol 73, No 2 August 1993
A 1 3 pM norepinephrine
'U %' C°02
10 % CC02 ° % CO2z
A
I1
z
0_
3k
z
00
wi
c.
E
5
4
-
-
2.5 pM norepinephrine
10 mM NH4CI
B
1
c
2
1
01
405
645k
330
1-
495k
255
X
c
0
1-
+U
It
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180
1053 0L
1-
345F
195F
10 min
A C1
4L
D
'15 min
FIG 3. Recordings showing effects of changes in Pco2 (panel A) and NH4Cl (panel B) on tone and [Ca 2+]i in vessels activated
with norepinephrine. In panelA, tone was induced by 3 uM norepinephrine, and Pco2 was changed. In panel B, the effects on tone
and [Ca 2+]i in a vessel stimulated with 2.5 ,uM norepinephrine by addition and removal of NH4Cl are shown.
changed by overflow vacuum suction in which the 3 -mL
chamber was washed with 50 mL of the new solution.
Measurement of Force and Membrane Potential
Simultaneous measurements of force and membrane
potential were made as previously described.17 In brief,
vessels were mounted in a myograph, and the internal
circumference was set to a normalized value as described above. Intracellular recordings of the membrane
potential were obtained using glass microelectrodes
filled with 3 M KCl. In these experiments, the solution
in the myograph was changed continuously (superfusion
with a peristaltic pump), and the change of solution was,
therefore, slower than in the experiments in which
fluorescence was measured. The effects of changes in
pHi on tone and membrane potential were assessed by
changing pHi with NH4Cl.
Solutions and Chemicals
PSS had the following composition (mM): NaCl, 119;
KCl, 4.7; KH2P04, 1.18; MgSO4, 1.17; NaHCO3, 25;
CaCl2, 2.5; EDTA, 0.026; and glucose, 5.5. The pH of this
solution was 7.45 to 7.50 when gassed with 5% C02-95%
02. Calcium-free PSS was made by adding 0.3 mM
EGTA to PSS not containing CaCl2. In experiments in
which Pco2 was changed between 10% and 0%, two
solutions were used. In the solution gassed with 10%
CO2-90% 02, NaHCO3 was increased to 50 mM (substitution of NaCl), and 5 mM HEPES was added. The pH
of this solution was titrated to 7.45 to 7.50. The solution
gassed with 100% 02 (0% Pco2) contained no NaHCO3
(substituted with NaCl), and 5 mM HEPES was added.
The pH of this solution was also titrated to 7.45 to 7.50.
Thus, in experiments in which Pco2 was changed, Po2 was
either 90% or 100%. Although high Po2 may affect the
arteries through generation of free oxygen radicals, we
consider it unlikely that a change between 90% and
100% will significantly affect the parameters studied.
Addition of 10 mM NH4Cl to the PSS and to PSS in
which potassium was increased (see below) was done
without substitution. In the solutions in which potassium
was increased, KCl was substituted with NaCl on an
equimolar basis to give the indicated potassium concentration, and 1 ,uM of the a-adrenoceptor blocker phentolamine (CIBA-GEIGY, Basel, Switzerland) was added
to avoid an effect due to release of endogenous norepinephrine. Stimulation with AIF4 was obtained by adding
5 mM NaF and 10 ,uM AICl3 to the PSS.
Other chemicals used were fura 2-AM, BCECF-AM,
and Pluronic F-127 (Molecular Probes, Inc, Eugene,
Ore). Ionomycin, PMA, ryanodine, caffeine, Cremophor EL, and norepinephrine-HCl were obtained from
Sigma Chemical Co, Poole, UK. Vasopressin was purchased from Sandoz, Basel, Switzerland.
Statistics
Data are shown as mean±SEM. Statistical significance of values was tested with a two-tailed paired
Student's t test and was considered significant at P<.05.
In fluorescence measurements, n indicates the number
of arteries, one artery per rat. In experiments in which
the membrane potential was measured, n indicates the
number of observations (always arteries from three rats
or more). Force is expressed as tension, which is the
measured force divided by two times the artery segment
length. In figures showing representative recordings,
[Ca 21]1 is shown as nanomolar concentration, whereas
averaged levels of [Ca2+]i shown in Tables 1 and 2 are
presented as pCaj (log[Ca 2]i, molar) values, because
[Ca2+]i values are log-normally distributed.'2
Jensen et al Effects of Changes in pH; on Tone
2.5 pM norepinephrine
A
I
2.5 pM norepinephrine
B
10 mM NHC1
317
10 mM NH.C1
0
0
-20
-20
p
U
-40
-40
-60
2
-60
m
5 m
.
M
2-
g
-1-
1-
0
O* *
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02
0
e
0-J
2.5 pM norepinephr-ine
C
10 mM NH4C1
2.5 pM norepinephrine
D
0
~~~~~~~JKJ~~~~~~~~~~~~~~~
0
-20
-20
a
aa
-40
W
-60
-40
-60
3.6
5.4.
5 mm
a
3.6.
0
j_
g
0
0
5 min
1
2.4
1.2
1.8.
04
*1
0.
01
FIG 4. Recordings showing effects of NH4Cl on membrane potential (Em) and tone in vessels activated with norepinephrine. It
not possible to keep the electrode in a single cell through a complete sequence of addition and washout of NH4Cl. Recordings,
therefore, show representative tracings from different cells in different vessels. The relaxation caused by addition of NH4Cl was
associated with a repolarization of Em (panel A). The longer-term effect of addition of NH4Cl was a sudden rebound of tone that
was associated with a depolarization (panel B). Removal of NH4Cl caused a transient increase of tone and the oscillations in tone
disappeared, but there was no further depolarization associated with this increase of tone (panel C). After the increase of tone, the
vessel relaxed, and Em repolarized (panels C and beginning of D). After approximately 15 minutes, the vessel developed tone again,
and this was associated with depolarization (panel D). After removal ofnorepinephrine, Em repolarized (panel D). Arrows in panel
C indicate where the electrode was impaled.
was
Results
Effects of Pco2 and NH4 Cl on pHi
Figs 1A and 1B show the effects of changing the Pco2
between 10% and 0% and the addition and removal of
10 mM NH,Cl, respectively, on pHi in vessels activated
with norepinephrine. In both situations, pH, was main-
tained at 7.45 to 7.50. Steady-state pHi in 10% Pco2 was
comparable to the level in 5% Pco2. Reducing Pco2
from 10% to 0% caused an increase in pHi of 0.48+±0.02
pH units (n=6) within 1 to 2 minutes. Thereafter, pHi
fell and stabilized at a pH value that was 0.13+±0.02
(n=6) pH units higher than the steady-state pHi in 10%
318
Circulation Research Vol 73, No 2 August 1993
TABLE 1. Effect of Norepinephrine, Potassium, AIF4, and Phorbol 12-Myristate 13-Acetate on Force Membrane Potential and pCaj
(-log[Ca211;) in Rat Mesenteric Resistance Arteries
Potassium (50 mM)
PMA (1 ,uM)
Norepinephrine (2-3 ,uM)
AIF4 (10 gM)
2.5±0.2 (17)
2.5±0.2 (14)
Tension (N/m)
1.1+0.1 (14)
3.7+0.3 (13)
6.39±0.03
6.34±0.04 (10)
6.38±0.04 (8)
6.59±0.05 (11)
(12)
pCaj
1.16±0.11 (8)
1.08±0.08 (11)
0.96±0.09 (12)
0.41±0.06 (10)
Tension/log([Ca2+],, nM)
25±3
32±2
27±3
AEm (mV)
(5)
13+3 (3)
(4)
(5)
PMA, phorbol 12-myristate 13-acetate; Em, membrane potential. Values are mean+SEM.
Tension and pCa, were measured during stimulation when steady state was achieved. L Em indicates the difference between Em during
stimulation and Em before stimulation of the vessel. Numbers in parentheses indicate the number of arteries (one artery per rat) or, in the
case of Em, the number of observations.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Pco2. When Pco2 was increased from 0% to 10%, pHi
fell 0.49±0.04 pH units (n=6) within 1 to 2 minutes;
after which, pHi recovered. Addition of 10 mM NH4Cl
caused an acute increase in pH, within 1 minute, which
averaged 0.44+0.04 pH units (n=4), followed by a
recovery of pHi to the baseline (Fig 1B). Washout of
NH4Cl now caused an acidification of 0.48±0.04 pH
units (n=4), followed by recovery of pHi to the baseline.
This pattern was also seen in vessels activated by
potassium, AIF4, and PMA and in resting vessels (data
not shown), although in resting vessels pHi in 0% Pco2
stabilized at a level 0.26±0.02 pH units (n=6) above the
level in 10% Pco2.
Effect of Changes in pHI, on Tone, [Ca 2+]i, and
Membrane Potential in Resting Vessels
Changes in pHi induced by either NH4C1 (Fig 2) or
changes in Pco2 (not shown) in resting vessels did not
markedly affect [Ca 2+]i or tension, although occasionally
a small transient increase in [Ca 2±]i was seen when
NH4Cl was washed out (Fig 2). Furthermore, the membrane potential was unaffected by the changes in pH,
(not shown) as reported previously.18
Effects of Changes in pH, on Tone, [Ca 2+]`,
and Membrane Potential in Vessels
Activated With Agonist
Submaximal activation with norepinephrine (2 to 3
,M, eliciting 70% to 90% of the maximal force) induced
force, an increase in [Ca 2±]i, and membrane depolarization (Figs 3 and 4, Table 1). Alkalinization induced by
changes in Pco2 or by NH4Cl induced a transient
depression of the norepinephrine-induced tone and a
transient decrease of [Ca 2±]i (Fig 3, Table 2). Acidification induced by Pco2 and by removal of NH4Cl was
associated with a transient increase of tone (acute
effect), along with transient increases in [Ca2"]i (Fig 3,
Table 2). After these transients, acidification induced by
Pco2 was associated with normalization or a small
sustained decrease of tone, although [Ca2+]i often remained elevated, whereas acidification after washout of
NH4Cl was accompanied by a decrease in tone and
[Ca 2+]i. However, 12 to 15 minutes after washout of
NH4Cl, when pHi was fully recovered, force and [Ca 2+]i
suddenly increased again. In two of eight experiments,
the force development preceded the increase in [Ca2+
(Fig 3B). We have no explanation for this. In norepinephrine-activated vessels, NH4Cl induced changes in
the membrane potential (Fig 4), which could explain the
changes in tone (ie, relaxation was associated with
repolarization, and force development was associated
with depolarization) except during the acute increase in
tension caused by acidification, when no change in
membrane potential was seen (Table 3). In vessels
submaximally activated with 0.5 U/L vasopressin, NH4Cl
had similar effects on force and membrane potential
(Table 3).
In four experiments, ryanodine was used to deplete
intracellular calcium stores. In these experiments, the
vessels were first exposed to 25 mM caffeine, which
produced a small transient contraction and a transient
increase in [Ca 2+]i (not shown). Thereafter, 10 ,M
ryanodine was added, which had no effect on [Ca 2+]i.
Ten minutes later, 25 mM caffeine was added, and now
no effect on tone and [Ca2+]i was seen (not shown). In
the continuous presence of ryanodine, tone was induced
with norepinephrine (Fig 5). The norepinephrine response (force and [Ca2+]_) were now unstable (Fig 5).
The effect of changes in pHi induced by NH4Cl on tone
and [Ca2+]i was, however, qualitatively unaffected by the
TABLE 2. Acute Effects of Changes in pH; on Tension and pCa; (-log[Ca2+]i) in Activated Vessels
Norepinephrine
Potassium
AIF4
PMA
A1Tension
ATension
ATension
ATension
ApCaj
ApCaj
ApCaj
ApCaj
+NH4Cl -2.6+0.3* +0.61±0.08* (4) -0.04±0.08 0.00±0.01 (4) -2.3±0.3* +0.29±0.05* (4) -0.12±0.03* +0.09±0.03* (5)
0% CO2 -2.5±0.3* +0.55±0.08* (4) +0.2±0.1 +0.16±0.03* (5) -2.0+0.3* +0.31±0.08* (4) -0.5±0.2
+0.13+0.01* (4)
-NH4Cl +2.7+0.3* -0.22+0.03* (4) +0.4+0.1* -0.09+0.01* (4) +2.0+0.5* -0.18+0.04* (4) +0.5+0.1* -0.17+0.05* (6)
10% CO2 +3.3+0.1* -0.13±0.01* (4) +0.6±0.2* -0.09±0.02* (5) +1.8±0.4* -0.15+0.01* (4) +0.6+0.1* -0.20±0.01* (4)
PMA, phorbol 12-myristate 13-acetate. Values are mean±SEM.
ATension is the maximal difference between the tension during the acute phase after the change in pHi and the tension before the changes
in pH,. ApCaj (-log[Ca2+J,, molar) represents the difference between the pCa, values corresponding to the tension values used to estimate
Atension. Concentrations of norepinephrine, potassium, AIF4, and PMA are as shown in Table 1. Numbers in parentheses indicate the
number of arteries, one artery per rat.
*Significantly different from zero at P<.05.
Jensen et al Effects of Changes in pH; on Tone
TABLE 3. Effect of Changes in pH; Induced by NH4Cl on
Membrane Potential in Vessels Activated With Norepinephrine
or Vasopressin
Membrane potential (mV)
Vasopressin
Norepinephrine
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NH4Cl addition
-63±4 (5)
-53±6 (3)
a) Acute
-27±2 (3)
-35±3 (5)
b) Long-term
NH4C1 washout
-23±2 (3)
-28±3 (4)
c) Acute
-58±1 (3)
-62±4 (5)
d) Long-term 1
-39±2 (5)
-34±3 (3)
e) Long-term 2
Values are mean±SEM.
Membrane potential measurements were taken (a) immediately
before tension started to increase again after the acute effect of
NH4Cl addition, (b) immediately before washout of NH4Cl, (c) at
maximal peak in tone after washout of NH4Cl, (d) during relaxation after washout of NH4Cl (long-term 1), and (e) after rebound
of force 12 to 15 minutes after washout of NH4Cl (long-term 2,
immediately before washing out the agonist). Numbers in parentheses indicate the number of observations (arteries from three
rats or more). The membrane potential in resting vessels before
activation was -60±2 mV (eight arteries). The membrane potential during activation with norepinephrine (2 to 3 ,uM) or vasopressin (0.5 U/L) before the first change in pH, was -36±4 mV
(five arteries) and -35±3 mV (three arteries), respectively.
presence of ryanodine (Fig 5). Additional experiments
were made to assess the effect of 10 ,uM ryanodine on
the transient contractile response and rise in [Ca2]i
induced by 10 ,uM norepinephrine in calcium-free PSS.
In three experiments, we found that ryanodine eliminated the transient responses to norepinephrine, sug-
gesting that the norepinephrine-sensitive calcium stores
were eliminated by this treatment.
10 pM ryanodine
3 pM norepinephrine
1O mM NH4CI
Z1.5 -, ! 1 {t!
2.0
g1.0
0.5
0.0
540
Z' 3 85-
319
Effects of Changes in pHI on Tone, [Ca 2+i,
and Membrane Potential in Vessels
Activated With Potassium
Stimulation with 50 mM potassium caused force
development, a sustained increase in [Ca'+]i (Fig 6), and
depolarization, as previously reported9J7 (Fig 7A, Table
1). In contrast to the acute effect of alkalinization on
tone in vessels activated with norepinephrine, the acute
effect of alkalinization on tone and [Ca'+]I was small
(Fig 6A, Table 2). Membrane potential was not affected
by alkalinization (Fig 7A). However, the acute effect of
acidification (Fig 6) was a transient force development
and a rise in [Ca 2]i (Table 2) without a change in the
membrane potential (Fig 7A), as seen during norepinephrine activation. Effects of NH4Cl on membrane
potential and force were also assessed in vessels exposed to 20, 30, and 40 mM potassium. In Fig 7B, a
recording from a vessel depolarized with 20 mM potassium is shown. Whereas increasing concentrations of
potassium caused a concentration-dependent depolarization, addition and washout of NH4Cl had small
effects on membrane potential as in vessels exposed to
50 mM potassium.
Effects of Changes in pHI, on Tone, [Ca 2+j, and
Membrane Potential in Vessels Activated With AlF4
The presence of 10 ,uM AF4- caused a force development that started after a delay of 2 to 10 minutes and
that was associated with a simultaneous increase in
[Ca2+]i (Fig 8, Table 1) and membrane depolarization
(Table 1). The ratio between force and [Ca 2]i during
steady-state activation with AF4- was similar to that
seen during activation with norepinephrine and higher
than that seen during activation with potassium (Table
1). The effect of changes in Pco2 (Fig 8A) and addition
and removal of NH4Cl (Fig 8B) on [Ca21]i and tone in
vessels activated with AF4- was similar to that seen in
vessels activated with norepinephrine (Table 2). In the
experiments in which membrane potential was measured, addition of NH4Cl failed to affect tone and
membrane potential markedly, probably because
smaller transient changes in pHi may be induced by the
slow change of solution in these experiments, although
removal of NH4Cl also caused potentiation of tone but
no change in membrane potential (not shown). The
long-term effect of acidification was, as in vessels activated with norepinephrine, a transient decrease in tone
and a transient repolarization of membrane potential
(not shown).
Effects of Changes in pH, on [Ca 2+li and Membrane
Potential in Vessels Activated With Phorbol Ester
Addition of 1 ,uM PMA caused a slow increase in
230
7515 min
FIG 5. Recordings showing effect of ryanodine on the
NH4Cl-induced changes in [Ca2+/i and tone of vessels activated with norepinephrine. Intracellular caffeine-sensitive calcium stores were depleted with ryanodine (not shown), and
tone was induced with 3 ,uM norepinephrine. NH4Cl was
added and was washed out 15 minutes later.
[Ca21]i and tension over 10 to 30 minutes after a delay of
5 to 15 minutes (Fig 9B). The increase in force was
associated with depolarization (Table 1). The acute
effects of alkalinization were small. Addition of NH4C1
caused a small decrease in [Ca 2]i and tone (Fig 9B,
Table 2), whereas changing Pco2 from 10% to 0%
caused a decrease of [Ca 2+]j while tone was little
affected (Fig 9A, Table 2). Acidification was associated
with a transient force development and a transient
increase in [Ca2"]j, as in vessels activated by other
means (Table 2), and the long-term effect of acidifica-
320
Circulation Research Vol 73, No 2 August 1993
A
~-50 mM potassium
10 %C02
B
--
10 %C02
0 %C02
i50 mM potassium
1.0
NH4C1
10 mM
1-11
-E
1.0 F
z
0
.2rn
0.5
c
0r
0:1
ala. 0.5 F
0.0
495-
420-
345-~4
3~~~~~~~~~~45-
1-
X~~~~~~5m
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Recording shwn fet
fcage nPu
pnlA
n
L1~
10min
FIG 6.
NH4Cl (panel B) and
activated with potassium.
tion
normalization of tone when acidification
was a
induced
by PCo2,
moval of
NH4Cl
caused
NH4C1
Effects of
was
whereas acidification induced
on
rable to the effects
transient relaxation
a
membrane
during
tone
potential
by re(Fig 9B).
were
induced with
compa-
AIF4
(not
shown).
on tone and
[Ca 2+j in vessels
Effects of Norepinephrine, Potassium, AlE4, and
PMA
In
with
a
on
Force, [Ca 2+j] and Membrane Potential
previous study, we have shown that tone induced
norepinephrine and potassium was associated with
a
sustained increase of
[Ca2"]i
in rat mesenteric small
arteries.9 As discussed,9 activation with
agonists gives, in
preparations, transient increases in
is well
[Ca2"]j, whereas in other preparations,
maintained during the contraction. In the present study,
some
Discussion
[Ca2"Ii
The purpose of this
study was twofold: (1) to assess
the effect of norepinephrine, high potassium, AIF4, and
PMA on force, [Ca 211i, and membrane potential in
small arteries and (2) to investigate the mechanisms
responsible for the acute effects of changes in pHi on
tone of rat mesenteric small arteries.
a
50 MM
vascular
we
[Ca"2I]
confirmed the sustained nature of the
during
stimulation
with
norepinephrine
demonstrated that stimulation with
ester was
also associated with
force and
[Ca"2+]
a
AIF4
rise
and
further
and
phorbol
sustained increase in
in rat mesenteric small arteries.
potassium
20 mM
potassium
A
A10 mm NH4C1
B
10 mM
NH.C1
0
0
-20
A-
-40
-40.
-60
-
10
a
mini
-60-J
1.4
10 miii
0.7f
a
9:
07
0
FIG 7.
Recordings showing effects of NH4El on tone and membrane potential (Em) in vessels activated with potassium. Vessels
depolarized by 50 mMpotassium (panel A) or 20 mMpotassium (panel B), and NH4Cl was added and was washed out 15
minutes later. In panel
recordings obtained in two different cells in the same vessel when adding and washing out NH4C1,
respectively, are shown. Arrows in panel B indicate where the electrode was impaled.
were
Jensen et al Effects of Changes in pH1 on Tone
A
AIF4 (NaF + AIC13)
321
A1F4 (NaF + AiC13)
F
B
10 %
10 mM NH4CI
-E
z
-E
Cu
c
C.
0.
420
345
270
195
120
v~
C-
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
45
15 min
45L
with 10 puM AF4 Tone
.
was
15 min
and [Ca 2+]i during tone induced
induced by washing with physiological salt solution containing 5 mM NaF and 10 uMMA1C13.
FIG 8. Recordings showing effects of changes in Pco2 (panel A) and NH4Cl (panel B)
AF4- is a known activator of G proteins,'9 although it
has also been shown to have an inhibitory effect on
cation transport ATPases.20 The AlF4 -induced depolarization, which was similar to that seen with norepinephrine, may suggest that G protein activation plays a role in
membrane depolarization and in the consequent maintained rise in [Ca 2]i during stimulation with agonists.
Himpens et a121 have reported an increase in [Ca21]i and
force after application of AIF4- in guinea pig ileal smooth
muscle in which the response was inhibited by calcium
antagonists. This is consistent with an important role for
calcium flux through potential-operated calcium channels during stimulation with AF4- in that preparation,
although the authors also stressed the potential importance of inhibition of the Ca 2-ATPase for the response.
The increase in [Ca2]i induced by phorbol ester,
which is believed to activate protein kinase C and, like
AF4-, bypasses the agonist receptor binding step, is in
agreement with a previous report22 although in contrast
to other publications that suggest the development of
force without an increase in [Ca 2+]1.2324 This may suggest multiple effects of phorbol esters or differences in
the effects of phorbol esters between different preparations. However, it has previously been shown that
contractions induced by phorbol ester in rat mesenteric
small arteries are dependent on the presence of extracellular calcium and that contractions elicited by phorbol ester are inhibited by the calcium antagonist felodipine,25 suggesting that the increase in [Ca 2]i may be
due to influx of calcium through voltage-operated calcium channels. This interpretation was directly supported by the phorbol ester-induced depolarization
seen in the present study. This observation is also
consistent with the possibility that activation of protein
kinase C may have a direct effect on monovalent or
divalent ion channel activity and could be one way
on
tone
through which norepinephrine activation leads to membrane depolarization in these vessels.
With all types of activation, the increase in [Ca'+]i
and tone was associated with depolarization. This observation supports the suggestion that one of the key
events in excitation-contraction coupling in this preparation is depolarization, which leads to an increase in
[Ca"2]i due to increased calcium influx through voltageoperated calcium channels and consequently to force
development.9 However, it was also apparent that the
effectiveness of [Ca"2]i in producing tension varied with
the mode of activation (Table 1), with comparable ratios
of tension to pCaj during stimulation with norepinephrine, AF4-, and PMA that were higher than the ratio
seen during activation with potassium.
Effects of Changes in pH, on Tone, Membrane
Potential, and [Ca2+]i
In the present study, we induced changes in pHi by
changing Pco2 between 0% and 10% or by adding and
washing out 10 mM NH4Cl. In this way, we obtained pHi
transients of approximately 0.4 to 0.5 pH units, which
we found in preliminary experiments were associated
with consistent mechanical responses. In a previous
study7 in which Pco2 was changed between 5% and 0%
CO2 and 5% and 10% C02, we found qualitatively
similar mechanical responses. The acute effects on tone
of changes in pHi induced by NH4Cl or Pco2 were
comparable, which suggests that the acute mechanical
responses were reflecting changes in pHi. However,
differences exist in the longer-term effects of the two
maneuvers that may be caused by events unrelated to
pHi. For this reason, we will only discuss the immediate
effects of changes in pHi.
Effects in resting vessels. In resting vessels, there were
only minor effects of NH4Cl-induced changes in pHi on
322
Circulation Research Vol 73, No 2 August 1993
10 % CO2
A
0 % CO2
10 % CO2
[ -
2F
a
1-
0
1
an
OF
FIG 9. Recordings showing effects of
changes in Pco2 (panel A) and NH4C1
(panel B) on tone and [Ca 2+li during
tone induced with phorbol 12-myristate
13-acetate (PMA, phorbol ester). The
force development and increase in
[Ca 2+]i is not shown in panel A. Tone
and [Ca 2+]i increased slowly on addition of PMA (panel B). After washout
of PMA (indicated by arrows), tone
was maintained.
1-1
M.~
+4
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
345
195
1
15 min
1n mM NH,Cl
4- _
..1U D1MAN
BX~~~~~~~~1 pm
f1
Au
lav
E
z
r.
0
.GC
0
4.
o
945F
795645[
I
495F
345
19545
membrane potential and force, as previously reported.18
This finding is in accordance with the small effect of
NH4C1 on [Ca2+]i in resting vessels (Fig 2) and indicates
that there may only be minor direct effects of changes in
pHi on ionic conductance in this preparation at rest.
This interpretation was further supported by the lack of
effect of NH4C1 on the membrane potential of arteries
partly depolarized with 20, 30, or 40 mM potassium.
Previous reports have suggested that in cerebral arteries
from dogs, rats, and cats acidification induced by
changes in pH. or Pco2 have effects on membrane
potential, probably through an effect on potassium
conductance.26,27 However, in these studies pHi was not
measured, and it was not possible to conclude whether
the effects were due to changes in pHi, pH,, or something unrelated to pH.28,29
10 min
The lack of effect of NH4Cl on [Ca2i]i contrasts with
previous report30 in which addition of NH4Cl caused a
transient increase in [Ca2+]i in A7r5 cells (a vascular
smooth muscle cell line). This may suggest a difference
in the effect of pHi in different vascular smooth muscles,
although it cannot be excluded that the effect in A7r5
cells could relate to the culture conditions.
Acute effects of acidification in activated vessels. The
acute effect of acidification was a potentiation of tone
irrespective of the mode of activation. Although the
increase in [Ca2"], may explain the increase in tone, it
was clearly not caused by depolarization. Two possibilities may explain the increase in [Ca2+]i and tone.
The first possibility is a displacement of loosely bound
calcium from intracellular calcium pools by the ina
Jensen et al Effects of Changes in pH; on Tone
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
creased proton concentration, ie, Ca2'-H+ competition
for intracellular binding places, or direct effects of pHi
on intracellular calcium stores causing a net release of
calcium. In a previous study,7 we provided evidence
consistent with proton-induced release of calcium from
an intracellular site. In the present study, we assessed
the possibility that this site is the norepinephrinesensitive calcium store by pretreating the vessels with
ryanodine. After ryanodine, the phasic rise in [Ca2+]
and force associated with acidification was still seen,
suggesting that the transient increase in [Ca 2+] was not
caused by release from a norepinephrine-, ryanodine-,
or caffeine-sensitive store. Interestingly, it has been
reported that the large pool of calcium, which binds to
the plasma membrane, is reduced by low pH,31 probably
because of competition between Ca2' and H+. If the
same is valid for the vascular smooth muscle studied
here, this sarcolemmal pool could be a potential source
of calcium during acute acidification.
The second possibility is an effect of low pHi on
transmembranal calcium flux. Recently, it has been
reported that the transient contraction associated with
the removal of NH4C1 is nifedipine sensitive in canine
pulmonary arterial smooth muscle32 and verapamil and
diltiazem sensitive in rat mesenteric resistance arteries.33 Thus, in both studies it was proposed that acidification affected the L-type calcium channels. In the
present study, the measurements showed that the membrane potential was unaffected by the acidification.
Hence, if acidification affects L-type calcium channels,
it must be through a modulation of the potential sensitivity of this channel; ie, the opening probability of the
channels increase for a given membrane potential.
However, this possibility is not consistent with an inhibitory effect of protons on L-type calcium channels in
isolated ventricular cells from guinea pig.34,35
Acidification has been suggested in cardiac muscle to
increase [Ca2+]i through increased Na+-H+ exchange,
leading to increased sodium and consequent net influx of
calcium through Na+-Ca2' exchange.36,37 A similar scheme
is probably unlikely in rat mesenteric small arteries because the Na+-Ca2' exchange seems to play a minor
functional role3839 and inhibition of the acid-induced
increase in sodium by omission of HCO3 (to inhibit
Na+-HCO- cotransport) and addition of amiloride (to
inhibit Na+-H+ exchange) potentiate the force induced by
acidosis.10
Acute effects of alkalinization in activated vessels. Relaxation induced by acute alkalinization was mainly seen
during activation with norepinephrine and AIF4; vessels
stimulated with phorbol ester or by increased potassium
were little affected. This was in contrast to the acute
effect of acidification, which caused force development
and an increase in [Ca 2+]i with all types of activation.
The relaxation was associated with repolarization of the
membrane. These observations suggest that the repolarization and consequent fall in [Ca 2+]i could be responsible for the relaxation caused by alkalinization of
tone induced with norepinephrine. Since a small fall in
[Ca2+]i was also seen during alkalinization in vessels
activated with potassium, in which no change in membrane potential could be detected, it cannot be excluded
that the alkalinization may also cause reduction in
[Ca2"]i through an additional mechanism, eg, increased
uptake of calcium into an internal store, increased
323
intracellular buffering of calcium, or increased extrusion
of calcium. In addition to this, it seems likely that an
increase in the effectiveness of calcium for force production is seen, since a small fall in [Ca2"]i was often
seen at a time when force was not reduced or even
increased (Fig 5B) during the alkaline phase.
The effect of alkalinization on vessels activated with
norepinephrine is most likely due to the inhibition of a
relatively proximal step in the excitation-contraction
coupling, since it was not found with high potassium.
Because a marked relaxant effect of alkalinization was
also seen in vessels activated with vasopressin and with
AIF4, it is unlikely that it represents a decreased binding
of norepinephrine to the receptor. Furthermore, because marked effects were seen in vessels activated with
AIF4 and less pronounced effects were seen in vessels
activated with phorbol ester, the step(s) sensitive to
alkalinization may be distal to activation of G proteins
but proximal to the activated protein kinase C.
Conclusion
In rat mesenteric small arteries, tone induced with
either norepinephrine, potassium, A1F4, or PMA was
associated with depolarization and an increase in
[Ca 2+]. However, the ratio of tension to [Ca 2+] varied
with the four types of activation, being highest with
norepinephrine, AIF4, and PMA and lowest with potassium. Tone induced with agonists was highly sensitive to
changes in pHi. Increases in pHi acutely relaxed agonistactivated vessels, which could be explained by repolarization and a consequent reduction in [Ca2"]i. The
underlying mechanism may reflect inhibition of the
step(s) relatively proximal in the excitation-contraction
coupling. The transient force development seen as a
result of acute acidification was probably caused by the
concomitant increase in [Ca2+]i, although it was not
consequent to depolarization. Release of intracellular
loosely bound calcium ions or release of calcium from
an intracellular calcium store (which is not norepinephrine, ryanodine, or caffeine sensitive) could play a role
in this response.
Acknowledgments
This study was supported by the Danish Medical Research
Council and the Novo Foundation. We thank Dr M.J. Mulvany, Institute of Pharmacology, University of Aarhus, for
valuable discussions.
References
1. Kontos HA, Richardson DW, Patterson JL. Effects of hypercapnia
in human forearm blood vessels. Am JPhysiol. 1967;212:1070-1080.
2. Daugherty RM, Scott JB, Dabney JM, Haddy FJ. Local effects of
02 and CO2 on limb, renal, and coronary vascular resistances. Am
J Physiol. 1967;213:1102-1110.
3. Ighoroje Ed, Spurway NC. Procedures to acidify cytoplasm raise
the tone of isolated (rabbit ear) blood vessels. J Physiol (Lond).
1984;357:105P. Abstract.
4. Furtado MR. Effect of NH4Cl on the contractility of isolated
vascular smooth muscle. Life Sci. 1987;41:95-102.
5. Aalkjxr C, Mulvany MJ. Effect of changes in intracellular pH on
the contractility of rat resistance vessels. Prog Biochem Pharmacol.
1988;23:150-158.
6. Feletou M, Harker CT, Komori K, Shepherd JT, Vanhoutte PM.
Ammonium ions cause relaxation of isolated canine arteries.
J Pharmacol Exp Ther. 1989;251:82- 89.
7. Nielsen H, Aalkjxr C, Mulvany MJ. Differential contractile effects
of changes in carbon dioxide tension on rat mesenteric resistance
arteries precontracted with noradrenaline. Pflugers Arch. 1991;419:
51-56.
324
Circulation Research Vol 73, No 2 August 1993
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
8. Danthuluri NR, Deth RC. Effects of intracellular alkalinization on
resting and agonist-induced vascular tone. Am J Physiol. 1989;256:
H867-H875.
9. Jensen PE, Mulvany MJ, Aalkjxr C. Endogenous and exogenous
agonist-induced changes in the coupling between [Ca 2+]j and force
in rat resistance arteries. Pflugers Arch. 1992;420:526-543.
10. Aalkjxer C, Cragoe EJ Jr. Intracellular pH regulation in resting and
contracting segments of rat mesenteric resistance vessels. J Physiol
(Lond). 1988;402:391-410.
11. Mulvany M, Halpern W. Contractile properties of small arterial
resistance vessels in spontaneously hypertensive and normotensive
rats. Circ Res. 1977;41:19-26.
12. Jensen PE, Mulvany MJ, Aalkjer C, Nilsson H, Yamaguchi H.
Free cytosolic calcium measured with calcium-selective electrodes
and fura-2 in rat mesenteric resistance arteries. Am J PhysioL In
press.
13. Lattanzio FA. The effects of pH and temperature on fluorescent
calcium indicators as determined with chelex-100 and EDTA
buffer systems. Biochem Biophys Res Commun. 1990;171:102-108.
14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca 2+
indicators with greatly improved fluorescence properties. J Biol
Chem. 1985;260:3440-3450.
15. Shorte SL, Collingridge GL, Randall AD, Chapell JB, Schofield
JG. Ammonium ions mobilize calcium from an internal pool which
is insensitive to THR and ionomycin in bovine anterior pituitary
cells. Cell Calcium. 1991;12:301-312.
16. Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular
pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979;18:2210-2218.
17. Mulvany MJ, Nilsson H, Flatman JA. Role of membrane potential
in the response to exogenous noradrenaline stimulation. J Physiol
(Lond). 1982;332:363-373.
18. Aalkjxr C, Hughes A. Chloride and bicarbonate transport in rat
resistance arteries. J Physiol (Lond). 1991;436:57-73.
19. Zeng YY, Benishin CG, Pang PKT. Guanine nucleotide binding
proteins may modulate gating of calcium channels in vascular
smooth muscle, I: studies with fluoride. J Pharmacol Exp Ther.
1989;250:343-351.
20. Missiaen L, Wuytack F, De Smedt H, Vrolix M, Casteels R. AIF4
reversibly inhibits P-type cation-transport ATPases, possibly by
interacting with the phosphate-binding site of the ATPase. Biochem J. 1988;253:827-833.
21. Himpens B, Missiaen L, Droogmans G, Casteels R. AIF4 induces
Ca 2+ oscillations in guinea-pig ileal smooth muscle. Pflugers Arch.
1991;417:645-650.
22. Rembold CM, Murphy RA. [Ca 2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction.
Am J Physiol. 1988;255:C719-C723.
23. Jiang MJ, Morgan KG. Intracellular calcium levels in phorbol
ester-induced contractions of vascular muscle. Am J Physiol. 1987;
253:H1365 -H1371.
24. Nishimura J, Khalil RA, Drenth JP, van Breemen C. Evidence for
increased myofilament Ca 2+ sensitivity in norepinephrine-activated
vascular smooth muscle. Am J Physiol. 1990;259:H2-H8.
25. Boonen HCM, De Mey JGR. G-proteins are involved in contractile responses of isolated mesenteric resistance arteries to agonists.
Naunyn Schmiedebergs Arch PharmacoL 1990;342:462-468.
26. Harder DR. Effect of H' and elevated pCO2 on membrane electrical properties of rat cerebral arteries. Pflugers Arch. 1982;394:
182-185.
27. Siegel G, Kampe C, Ebeling BJ. pH-dependent myogenic control
in cerebral vascular smooth muscle. In: Cerv6s-Navarro J,
Fritschka E, eds. Cerebral Microcirculation and Metabolism. New
York, NY: Raven Press, Publishers; 1981:213-226.
28. Smeda JS, Lombard JH, Madden JA, Harder DR. The effect of
alkaline pH and transmural pressure on arterial constriction and
membrane potential of hypertensive cerebral arteries. Pflugers
Arch. 1987;408:239-242.
29. Harder DR, Madden JA. Cellular mechanism of force development in cat middle cerebral artery by reduced PCO,. Pflugers Arch.
1985;403:402-404.
30. Siskind MS, McCoy CE, Chobanian A, Schwartz JH. Regulation of
intracellular calcium by cell pH in vascular smooth muscle cells.
Am J Physiol. 1989;256:C234-C240.
31. Grover AK, Crankshaw J, Triggle CR, Daniel EE. Nature of
norepinephrine Ca-pool in rabbit aortic smooth muscle: effect of
pH. Life Sci. 1983;32:1553-1558.
32. Krampetz IK, Rhoades RA. Intracellular pH: effect on pulmonary
arterial smooth muscle. Am J Physiol. 1991;260:L516-L521.
33. Matthews JG, Graves JE, Poston L. Relationship between pH, and
tension in isolated rat mesenteric resistance arteries. J Vasc Res.
1992;29:330 -340.
34. Irisawa H, Sato R. Intra- and extracellular actions of protons on
the calcium current of isolated guinea pig ventricular cells. Circ
Res. 1986;59:348-355.
35. Kaibara M, Kameyama M. Inhibition of the calcium channel by
intracellular protons in single ventricular myocytes of the guineapig. J Physiol (Lond). 1988;403:621-640.
36. Bountra C, Vaughan-Jones RD. Effect of intracellular and extracellular pH on contraction in isolated, mammalian cardiac muscle.
J Physiol (Lond). 1989;418:163-187.
37. Kim D, Cragoe EJ Jr, Smith TW. Relations among sodium pump
inhibition, Na-Ca and Na-H exchange activities, and Ca-H interaction in cultured chick heart cells. Circ Res. 1987;60:185-193.
38. Mulvany MJ, Aalkjxr C, Petersen TT. Intracellular sodium, membrane potential and contractility of rat mesenteric small arteries.
Circ Res. 1984;54:740-749.
39. Mulvany MJ, Aalkjxr C, Jensen PE. Sodium-calcium exchange in
vascular smooth muscle. Ann N YAcad Sci. 1992;639:498-504.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Force, membrane potential, and [Ca2+]i during activation of rat mesenteric small arteries
with norepinephrine, potassium, aluminum fluoride, and phorbol ester. Effects of changes
in pHi.
P E Jensen, A Hughes, H C Boonen and C Aalkjaer
Circ Res. 1993;73:314-324
doi: 10.1161/01.RES.73.2.314
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