Role of Calcium in Contractile Excitation of
Vascular Smooth Muscle by Epinephrine
and Potassium
By William H. Waugh, M.D.
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• Considerable evidence suggests that in vertebrate skeletal muscle,1"6 cardiac muscle,7"10
and visceral smooth muscle,11"17 calcium may
be the agent which links membrane excitation
to myoplasmic contraction. However, the evidence is meager that calcium may be the agent
in excitation-contraction coupling of blood
vessel muscle.3 8~20 Since the smooth musculature of blood vessels is mainly of a multiunit
(motor) type rather than of a visceral (automatic) type,21-22 an investigation of its functional behavior separate from that of visceral
smooth muscle is required.
The role of calcium in excitation-contraction
responses of vascular smooth muscle was investigated in the following study of perfused
isolated small arteries of the dog. Excitation
was induced both by epinephrine and by excess potassium ion. The results extend a recent study of arterial smooth muscle in which
adrenergic neurohormone appeared to activate
contraction by some mechanism other than
primarily through membrane depolarization
or sodium or chloride influx.20
Methods
Segments of dog intestinal arteries (intramesenteric) 1.4- to 2.2-cm. long and of 0.5- to 1.0-mm.
external diameter were excised, stripped of adventitia, and perfused by a constant-flow technique,
as previously described.20 The constant-flow rate
used in various experiments was 0.75 to 0.90 ml./
min. Test doses of chemicals in volumes of 0.10
to 0.25 ml. were injected upstream to the perfusate
pump of constant flow. Smooth muscle contractile
From the Department of Medicine, University of
Kentucky College of Medicine Lexington, Kentucky.
Supported by Grants HB-06092 and HE-06347 from
the National Heart Institute, V. 8. Public Health
Service.
Pnrt of this work was reported in abstract form
in Fed. Proo. 21: 120, 1962.
Received for publication June 4, 1962.
Cirovlation Research, Volumo XI, December 2969
reactions of vasoconstriction were metered and
recorded on oscillographic paper, as previously
described,20 as elevations in perfusate pressure
immediately upstream from the arterial segment.
The test agents were (1) dZ-epinephrine HC1 in
5 per cent glucose containing 10"6 ascorbic ncid;
and (2) the following salts in isosmotic aqueous
solution, injected individually (mM/L.) : KC1,150;
NaCl, 150; Nal, 150; CaCl2, 100; Cal2, 100; and
MgCl2, 100.
Two previously described electrolyte-glucosecreatine solutions of pH 7.4 were used as the basic
perfusion fluids20; these were termed isosmotic
solution NaCl, 140 mM/L., and isosmotic solution
K2SO4, 96 mM/L. The two were identical in CaCl2
(L6 mM/L.), MgCl2 (0.7 mM/L.), glucose (5.5
mM/L.), and creatine (0.3 mM/L.) concentration.
They contained phosphate buffer (2.0 mM/L.)
added as sodium and potassium salts, respectively.
The solutions differed in that the former contained
4.0 niM KC1/L. and no K2SO4, while the latter
solution contained no NaCl or other sodium salt.
In some experiments, the basic perfusion fluids
were modified by the inclusion of ethylenediaminetetraacetate (EDTA), 10 mM/L., for the chelation
of divalent cations. The EDTA had been added in
the fully acidic form (H 4 EDTA). The EDTAeontaining solutions were used only after they had
been brought to pH 7.4 by the addition of NaOH
or KOH alone, or with either Ca(0H) 2 or
Mg(0H) 2 , 10 mM/L. At pH 7.4, the predominant salt species of EDTA was NaaHEDTA,
K8HEDTA, CaNa2EDTA, CaK2EDTA, MgNa2EDTA, or MgK2EDTA. These species were selected to describe the various 10-mM EDTA/L.
solutions employed. In such experiments, the control basic perfusion fluid contained sucrose, 40
mM/L., added to achieve isosmoticity.
Results
EDTA PREVENTION OF CONTRACTIONS
INDUCED BY EPINEPHRINE PERFUSION
During perfusion with simply the isosmotic
solution NaCl, 140 mM/L., the arterial segment was relaxed. When the perfusate was
abruptly changed from this control solution
to an identical solution with epinephrine
added to an effective concentration, vasocon927
928
ISOSMOT1C
WATJGH
PERFUSION
THROUGHOUT, No Cl
140 m M / L
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the development of the epinephrine-induced
contractions of the arterial muscle.
The observations with MgNa2EDTA and
CaNaoEDTA indicate that the prompt prevention by simultaneously perfused EDTA of the
epinephrine-induced contractions was apparently through concurrent removal of ionized
calcium (from the extracellular fluid or superficial parts of the smooth muscle cells), rather
than through removal of magnesium or
through a direct depressant effect of the
EDTA anion. The use of nonEDTA solution
made free of calcium ion simply by omission
of calcium salt in the preparation of the perfusate depressed more slowly the vasoconstriction from perfused epinephrine.
EDTA PREVENTION OF CONTRACTIONS
INDUCED BY POTASSIUM
FIGURE 1
EDTA prevention of vasoconstrictor pressure responses induced by epinephrine perfusion of arterlul segments. Constant flmo was maintained except
for the brief periods of stopped flow preceding
the arroio marks on these serial records (top to
bottom). At the arroio marks, the perfusate of
isosmotic solution NaCl, 140 mM/L., was changed,
to a similar erne except that it now contained added
epinephrine (2 X 10-6) by itself or with added
NdjHEDTA, MgNajEDTA, or CaNatEDTA (10
mM/L.).
stnotion developed promptly. The development of vasoconstriction was promptly largely
prevented, and the developing: vasoconstriction
was promptly abolished by including either
Na8HEDTA or MgNa2EDTA, 10 mM/L., in
the epinephrine-containing perfusate. These
findings are illustrated in the typical records
of figure 1. Simultaneously perfused CaNa^EDTA, 10 niM/L., completely failed to inhibit
Isosmotic solutions of high potassium sulfate concentration have been shown to depolarize completely visceral smooth muscle
cells.28'24 When the solution perfusing the
artery was abruptly changed from the isosmotic solution NaCl, 140 mM/L., to the isosmotic solution K2SO4, 96 mM/L., vasoconstriction immediately developed. Such responses
are illustrated in records 1A and C, 2A and
C, and 3A and C of figure 2. On the other
hand, when potassium depolarization of the
arterial segment was carried out by replacement of the sodium chloride—rich perfusate
with isosmotic solution containing both K0SO4,
96 mM/L., and KSHEDTA, 10 mM/L., the
immediate vasoconstriction response was much
reduced and very short-lived, as shown in
part IB of figure 2. Record 2B of figure 2
reveals that the contractile responses upon
perfusion with isosmotic solution K0SO4, 96
mM/L., were similarly promptly prevented by
inclusion of MgK2EDTA in the potassiumrich perfusate. The contractions were not in
the least prevented or reduced by use of the
EDTA in the form of CaTv.EDTA, 10 mM/L.,
as illustrated in part 3B of figure 2. Thus,
this EDTA prevention of contractions induced
by high extracellular concentrations of potassium or potassium depolarization apparently
resulted from concurrent removal of ionized
calcium from the artery.
Circulation Research', Volume XI, December lift
929
OALOITTM A N D VASOULAB SMOOTH MUBOLE
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CHWNC ID K^Q,
FIGURE
EDTA prevention of vasoconstrictor pressure responses induced by high potassium perfusion of arterial segments. At the arrmo marks on these serial records (1A to G, 2A to
C, 3A to C), the perfusate of isosmotic solution NaCl, 140 mM/L., was changed to the
isosmotic solution KSSO^, 96 mM/L., either without added EDTA or with added
K,HEDTA, MgKtEBTA, or CaKtEDTA (10 mM/L.).
EDTA REMOVAL OF POTASSIUM CONTRACTURE AND PREVENTION OF ADRENEROIC
CONTRACTIONS OF POTASSIUM
DEPOLARIZED MUSCLE
When perf usion of the artery was continued
for more than a few minutes with the isosmotic solution K2SO4, 96 mM/L., the vasoconstriction induced by the high potassium
concentration diminished gradually until a
relatively steady state of lessened vasoconstriction was reached. As previously described,20 during this sustained potassium
depolarization and contracture essentially
unimpaired contractions were induced by injections of epinephrine.
The potassium contracture was rapidly removed upon inclusion of KSHBDTA, 10
mM/L., in the potassium sulfate-rich perfusion medium. In this potassium-depolarized
muscle, contractile responses to injected epinephrine were promptly much diminished by
this inclusion of KRHEDTA in the perfusate;
they were practically or completely prevented
Circulation Research, Volume XI, December 190t
after several minutes of continued perfusion with this isosmotic solution containing
K3HEDTA, 10 mM/L. An experiment of this
kind is illustrated in figure 3.
Inclusion of MgK2EDTA, 10 mM/L., in the
perfusate similarly abolished the potassium
contracture and similarly prevented the epinephrine-induced contraction in the potassium-depolarized arterial muscle. Impairment
did not result when the perfusion fluid contained the EDTA in the form of CalwEDTA,
10 mM/L.; the basic calcium content of 1.6
mM/L. of this solution had not been complexed or deionized by the EDTA. The effect
of EDTA treatment as the magnesium complex and as the calcium complex on the epinephrine response is shown in figure 4.
Figure 4 also reveals more evidence that the
EDTA prevention of the epinephrine-induced
contractions of the previously depolarized
muscle was through calcium deionizing by the
EDTA. As illustrated in the lower record of
930
WATTGH
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t
I
inn
\r in
FIGURE 3
EDTA removal of potassium contracture and EDTA prevention of epinephrine-induced
contractions of potirssium-depolarized arterial muscle. In the upper record is shown a
lurge vasoconstrictor pressure response to 1 fxg. of epinephrine injected during potassium
contracture. In the lower record at the first arrow, potassium-rich perfusion ivas stopped
momentarily and restarted with modified solution containing EDTA—K3HEDTA (10
mM/L.) having been substituted for sucrose (40 mM/L.). Epinephrine injection was
then repeated.
figure 4, epinephrine contractions were immediately elicited Avhen calcium ion was injected
simultaneously with the epinephrine into the
perfusate stream of MgKoEDTA-containing
solution. The same amount of calcium ion
(e.g., 10 /JM. CaCl2) injected by itself caused
practically no contraction during perfusion
with the MgKoEDTA and K2SO4-rieh solution and none during control perfusion with
the isosrnotic solution NaCl, 140 mM/L. (see
also upper right record of fig. 4).
However, as shown in the main upper and
middle records of figure 4, injection of the
same amount of calcium chloride by itself
evoked contraction of the same artery during
perfusion with either the isosmotic solution
K2SO4, 96 mM/L., alone or with the inclusion
of the EDTA as CaK2EDTA in this highpotassium perfusate. These particular calcium-induced contractions were much smaller
in degree than those evoked by the combined
calcium ion—epinephrine injection in the
MgK2EDTA medium. In contrast to the re-
sult with the combined injection in the perfusion solution containing the MgKoEDTA,
in the "control" isosmotic solution K0SO4, 96
mM/L., alone and with added CaK2EDTA,
the combined injection of calcium ion and
epinephxine evoked no greater vasoconstriction than could be attributed to a mere summation of the injected calcium ion and
epinephrine-mduced contractions considered
separately.
EFFECT OF POTASSIUM DEPOLARIZATION
ON CALCIUM PERMEABILITY OF
ARTERIAL MUSCLE CELLS
Injection of isosmotie calcium chloride solution in a sizable volume, which was yet of
subthreshold contractile strength, during use
of the isosmotic solution NaCl, 140 mM/L.,
induced arterial muscle contractions during
sustained potassium depolarization produced
by use of the isosmotic solution K2SO4, 96
mM/L. With the use of each of these two perfusates, vasoconstriction failed to be evoked
by equivolumetric injections of isosmotic magCirculation Research, Volumo XI, December lQ6t
931
CALCIUM AND VA80TTLAB SMOOTH MUSCLE
„ . .PCTFU** WITH aOSMOTK SOL.K.jq, MaW/L ; 40 HM
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FIGURE 4
EDTA prevention of epinephrine contractile excitation in potassium-depolarized muscle
and its restoration by concurrent injection of calcium ion. Vasoconstrictor results of
both separate and combined bolus injections of 1 fxg. epinephrine and 10 /xM calcium
chloride are shown in various control records above and in the record during MgKtEDTA
treatment below (see text).
nesium chloride and isosmotic sodium chloride
solutions. Thus, the contractions induced by
injected calcium chloride solution appeared to
be the specific result of calcium ion injection.
Figure 5 illustrates the results of one experiment. This experiment was rather atypical in
that injected calcium chloride which did not
cause contraction of the polarized or resting
muscle caused, during sustained potassium
depolarization, an unusually large contractile
response; it was of similar magnitude to the
large response induced by 1 /ng. of injected
epinephrine.
Larger injections of isosmotic calcium chloride solution, which caused contractions during use of the sodium chloride-rich perfusate,
consistently evoked greater vasoconstriction
when administered to the potassium-depolarized muscle. However, the potassium depolarization did not appreciably change the magnitude of the vasoconstrictor pressure response
to injected epinephrine.
Circulation RfMeareh, Volume XI. December 19S1
The relative contractions induced by standard injected amounts of isosmotic calcium
chloride solution are a measure of changes in
the calcium influx and inward permeability to
calcium in these experiments if the isosmotic
calcium ion solution exerted its contractile
action not at the muscle cell membrane but
directly, by an intracellular effect on the contractile machinery. If this is so, it is demonstrated that potassium depolarization greatly
increases the permeability of the arterial muscle cells to calcium.
The thesis is proposed that the relative magnitude of the arterial muscle contractions
induced by injections of isosmotic calcium
chloride solutions indeed measured changes in
membrane permeability and influx of calcium
into the arterial muscle cells. The thesis is
strongly supported by the following facts:
(1) Calcium generally exerts a stabilizing
rather than depolarizing effect on cell membranes.28"27 (2) Potassium depolarization or
932
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cathodal current stimulation sufficiently increases the calcium permeability of skeletal
muscle so that calcium ions applied externally
as isosmotic calcium chloride solution penetrate into the cell interior to produce contraction28; the isosmotic calcium chloride solution
does not cause contraction of resting polarized
or uninjured skeletal muscle.2S'2U (3) Of all
the physiological ions (Na, K, Ca, Mg, Cl,
ATP, HPO4, citrate, and oxalate) which, in
small quantities, have been injected intracellularly or applied locally to bare myoplasm,
calcium alone causes contraction.28~aB (4)
During potassium depolarization of skeletal
muscle,4' ° cardiac muscle," and aortic smooth
muscle,18 there are increased transmembrane
fluxes of calcium. (5) In heart muscle, the
degree of potassium contracture varies with
extracellular concentration of calcium without an essential change in the potassium depolarization.7
EFFECT OF EPINEPHRINE EXCITATION ON
CALCIUM PERMEABILITY OF PREVIOUSLY
POLARIZED ARTERIAL MUSCLE
Because of the immediately foregoing results and considerations, the vasoconstrictor
effectiveness of acutely administered isosmotic
calcium salt presented itself as a method for
studying the mechanism of hormonally induced excitation-contraction of vascular
smooth muscle. Vasoconstrictor excitation by
adrenergic neurohormone was investigated by
employment of this method of testing calcium
permeability and calcium influx; this method
is, of course, predicated on the intactness of
.the contractile machinery of the muscle cells.
Figures 6 and 7 reveal that in the artery
perfused with the isosmotic solution NaCl,
140 mM/L/., injection of a barely contractile
dose of calcium chloride caused much greater
vasoconstriction following epinephrine excitation. The top and bottom records of figure 6
and the top record of figure 7 show that the
calcium effect was increased by epinephrine
even during, or shortly after, relaxation of
the epinephrine-iudueed contraction. The middle record of figure 6 illustrates that during
sustained epinephrine excitation and contraction (induced by perfusion of a constant con-
WATJOH
centration of epiuephrine) the acutely injected calcium ion effected very much greater
vasoconstriction than during the relaxation
phase of epinephrine contraction.
The lower record of figure 6 also indicates
that these epineplirine-increased contractions
from injected calcium were not due to the
actual myoplasmic contractions produced by
epinephrine, i.e., they were not a nonspecific
simple treppe, or staircase, of muscular contraction.80 Instead, this lower record of figure
6 apparently indicates that these epinephrineincreased contractions from injected calcium
were the result of increased calcium influx
arising because the permeability to calcium
was specifically increased by an action of epinephrine. A second injection of epinephrine,
identical in dosage to an epinephrine injection
given shortly before, evoked contraction of
similar magnitude before complete relaxation
of the preceding epinephrine-induced contraction. On the other hand, calcium, when given
similarly before complete relaxation of the
epinephrine-induced contraction, caused a far
greater contractile response than the pre-epinephrine response to injected calcium.
EFFECT OF EPINEPHRINE EXCITATION ON
CALCIUM PERMEABILITY OF
POTASSIUM-DEPOLARIZED
ARTERIAL MUSCLE
As shown in the upper and middle records
of figure 7, during use of the isosmotic solution NaCl, 140 niM/L-, the contractions arising from injected calcium ion were consistently much augmented both by excitatory
amounts of epinephrine and by excitatory
amounts of isosmotic potassium chloride solution injected shortly before the calcium injection. This similar calcium augmentation effect,
lasting a few minutes, and the fact that sustained potassium excitation also increased the
inward membrane permeability to calcium as
did sustained epinephrine perfusion (previously illustrated in figs. 4, 5, and 6, respectively) suggest that epinephrine excitation
increased the calcium permeability of the
arterial muscle membrane, perhaps, by means
of electrical depolarization analogous to potassium depolarization of the membrane.
Circulation Research, Volume XI, December 19BS
933
CALCIUM AND VASOULAB SMOOTH MUSCLE
_
HOJWIC
*OL ,N.CI HO HIM/1. ;7MW
IIOWOTIC
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10 MICH0K0LE5
SOL , K,»O, M m / I
M«CI,
: H
10 MIOKOMOLCS C«Ct,
FIGURE S
Effect of potassium depolarization on the muscular action of calcium ion injected in
high concentration. In the upper record are shown lack of vasoconstrictor pressure rises
after 0.1-ml. injections of isosmotic NaCl, CaCl,, and MgCls solutions and the pressure
response to epinephrine when the artery had been perfused toith the NaCl-rich solution.
In the lower record are shown the presstire elevations induced in the potassium-depolarized state by the same injected doses of GaClt and epintphrinp and the continued ineffectiveness of injected HlgClt.
However, this was apparently not the
essential mechanism. As shown in figure 8,
calcium ion injections which produced reproducible contractions of the muscle cells depolarized by perfusate use of the isosmotic
solution KoS04, 96 mM/L., induced much
greater contractions during epinephrine excitation of this muscle already depolarized by
the potassium sulfate—rich perfusion.
Paradoxically, isosmotic potassium chloride
solution injected during perfusion with the
isosmotic solution NaCl, 140 mM/L., caused
consistently transitory relaxation of the artery when it was much constricted by epinephrine; the potassium chloride injections constricted the resting vessel not treated with
epinephrine. This paradoxical relaxing effect
of a depolarizing amount of potassium is illustrated in the middle record of figure 6. Equivolumetric injection of isosmotic magnesium
chloride solution also had a relaxing effect on
the epinephrine-contracted muscle.
Circulation ReMearch. Volume XI, December 1962
EFFICT OF IODIDE I N INCREASING
CALCIUM-INDUCED CONTRACTIONS
OF ARTERIAL MUSCLE
Equimolar injections of isosmotic calcium
iodide solution produced contractile responses
of the artery perfused with the isosmotic solution NaCl, 140 mM/L., several times greater
than did injections of isosmotic calcium chloride solution. A typical result is shown in the
bottom record of figure 7. Equivolumetric
injections of isosmotic sodium iodide and isosmotic sodium chloride solutions failed to elicit
vasoconstrictor responses.
The greater contractions induced by calcium administered as the iodide salt over
those induced when calcium was injected as
the chloride salt may have been caused by a
transitory depolarization resulting from outward diffusion of chloride ion (with potassium
ion) when the extracellular chloride concentration was transiently reduced by iodide
replacement.25 It seems much more probable,
however, that the greater contractions were
934
WATTQH
I
E
cr
UJ
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FIOURI 6
Effect of epinephrine contractile excitation on calcium permeability of previously polarized arterial muscle. Pressure elevations induced by a standard injection of isosmotic
CaClf solution were used as an index of calcium influx and calcium permeability changes
induced by epinephrine (see text). In the upper and lower records, 1 (j.g. epinephrine
was injected as a bolus; in the middle of these serial records, epinephrine was perfused
at constant concentration (5 X 1O~7). The middle record also reveals vasodUatation from
isosmotic KCl and MgClt solutions injected during epinephrine-indticed vasoconstriction.
due to greater transmembranous diffusion of
calcium paired with iodide than with chloride,
since injected sodium iodide did not evoke
contraction. The findings suggest that transmembranous passage of calcium occurs significantly in the form of associated ion pairs of
calcium. The greater polarizability and lesser
hydration energy of iodide25 should facilitate
the transrnembranous diffusion of calcium in
an associated ion-pair form (a weakly hydrated state) when the anion is iodide rather
than chloride. The effective hydrated diameters of these two halides as the free anions are
the same, while the unhydrated diameter of
iodide is larger.25
Discussion
Epinephrine excitation of arterial muscle
contraction seems to be a mechanism which
primarily acts through some other process
than electrical depolarization of the membrane
or sodium or chloride influx, because the essential operation of epinephrine is unimpaired
in arterial muscle which has been completely
depolarized by the external application of
potassium sulfate in the virtual absence of
extracellular sodium and near absence of extracellular chloride.20 In the present investigation, it was shown that calcium ions are
essential for the induction of contractions by
epinephrine in the resting (polarized) muscle
as well as in the muscle depolarized by potassium sulfate solution devoid of sodium salt
and containing less than 5 rnM chloride/!/.
Since, in the calcium-depleted and potassiumdepolarized muscle, the contractions arising
from injected epinephrine were immediately
restored by simultaneous injection of calcium
ions, epinephrine excitation of vascular
Circulation Research, Volume XI, December list
935
CALCIUM AND VASOTTLAB SMOOTH MUSCLE
23 UICROWOUS
CiCL,
23
MiCffOMOLES
C*D,
x
E
E
Id
CO
CO
0.
23 utcmMOLES
25 MICROMOLES
Cad,
UJ
<
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23
UlCttOWOLES
Ccl,
23 MKROWOIE!
C«tt
riOURI 7
Effect of excitation from acutely injected epinephrine and potassium on calcium-induced
contractions of arterial muscle and the iodide-increased, calcium-induced contractions
of arterial muscle. 1A and IB are serial records (see text) shoioing, respectively, the
excitatory effects of epinephrine and of KCl injected as a bolus. Record 2 (bottom) is
from a different arterial perfusion experiment in which the vasoconstrictor effect of
equimolar injections of isosmotic CaCl, and Cal, solutions are compared.
smooth muscle seems to involve basically a
membrane reaction concerned with calcium.
Both excitatory concentrations of extracellular potassium and contractile-inducing
amounts of epinephrine increased the permeability to calcium and increased calcium flux
into the vascular smooth muscle fibers. These
similar actions make it appear that the increased calcium permeability and influx produced by epinephrine excitation might be
through electrical depolarization, since high
extracellular concentrations of potassium depolarize cell membranes. However, the increased calcium permeability of epinephrine
excitation was clearly not primarily produced
by electrical depolarization, because epinephrine consistently increased the calcium permeability and influx both in muscle depolarized by potassium sulf ate—rich solution devoid
of sodium and in muscle previously polarized.
Therefore, it appears that the primary essential process in vasoconstrictor excitation by
CiretUation Retearch, Volume XI, December l»tt
adrenergic neurohormones is a mechanism
which increases nonelectrically the cell permeability to calcium.
It is well documented that calcium exerts
a stabilizing effect on cell membranes, calcium
depletion of which leads to increased membrane permeability to various ions.25"27 Epinephrine and other adrenergic agents probably initiate the vasocontractile excitatory
process by pairing stereochemically with
phosphate anionic sites of the alpha-adrenergic
receptor of the cell membrane.37 It is suggested that the primary reception of epinephrine triggers a primary increased membrane
permeability to calcium and other ions by a
presently unknown mechanism; this, however,
may primarily consist of a local geometric
change in the phosphorylated receptor due to
charge redistribution. The latter may trigger
a primary release of membrane calcium with
dispersion of the normally arranged phospholipid structure of the plasma membrane.88
936
WAUGH
ISOSUOTtC SOL .KgSO,
MmM/L ,
r
to
30
0
£0 MICftOWOLCS Ccdt
tSOSMOTK SOL.KySO,
o a r cw
*
tO UICftOUOLE3
1 MN
CoO,
20 MK»0UOLCS
CaOt
HiM/L; 100 MN
to
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30
0
20 HCIKMUI
to utcjtowous c*a.
C*O,
FIGURE 8
Epinephrine-increased permeability to calcium in arterial muscle already depolarized
by high potassium sulfate perfusion (see text). The upper record shows reproducible
contractions (vasoconstrictor pressure responses) from a standard injection of isosmotic
CaClf solution. The middle and loiver serial records reveal that prior injection of a
small contractile dose of epinephrine (0.5 /ig.) greatly increased the contractile action
of injected Gu ion solution even in this potassium sulfate—depolarized muscle.
In this hypothesis, epinephrine in its basic
excitatory reaction of increased permeability
to calcium and other ions induces secondarily
electrical depolarization of vascular muscle
through movements of sodium, chloride, and
other ions. Epinephrine appears normally to
induce depolarization in its contractile excitation of resting or polarized vascular smooth
muscle.80'40 However, the amount of electrical
activity induced by epinephrine may be
slight.41
Concurrent depolarization should be important if there is to be electrical propagation
of the excitatory process to adjacent smooth
muscle cells. However, this propagation seems
to be of no quantitative significance in the
vascular contractions induced by Immorally
moA'ing epinephrine.20 On the other hand, the
epinephrine-induced depolarization with sodium influx would seem to be a prerequisite
for a normal rate of relaxation of epinephrine-induced contractions; sodium hastens the
relaxation-recovery process.20
It is not clear whether calcium ions of the
general extracellular fluid, at normal concen-
trations around 1.5 mM/L., move inward
sufficiently during excitation to contribute
significantly to the raised intracellular calcium content. The latter may arise solely or
largely from an influx of calcium released in
excitation from labile membrane stores. The
concentration of calcium in cell membranes is
much greater than that of the general extracellular fluid.42
The general hypothesis of membrane excitation of muscle is that the electrical depolarization process itself, which involves sodium,
potassium, and anion fluxes, is the normal
primary reaction of excitation which releases
membrane calcium as a link between excitation
and contraction. Contrary to this hypothesis
of Sandow,43 Csapo,8-1S Frank,6-44 Shanes,2
and others are the experiments of Robertson,14 Durbin and Jenkinson,10-45 and
others16-24'4(I'47 in potassium-depolarized visceral smooth muscle and the experiments of
this and previous investigations20'41'48 in potassium-depolarized vascular smooth muscle.
The vascular smooth muscle experiments may
be interpreted as evidence that adrenergic
Circulation Research, Volumo XI, Dacombor 190t
OAIiOnJM AND VASCULAR SMOOTH MUSCLE
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excitation of vascular smooth inusele is not
the result of a primary electrical depolarization process which produces increased calcium
release and increased membrane permeability
to calcium.
However, a state of electrical depolarization
may perhaps be essential for adrenergic stimulation of vascular smooth muscle contraction
to a normal magnitude; this may be because
of a facilitating effect of depolarization on the
association of ion pairs- 5 ' 40 for migration
through membrane regions of low effective
dielectric constant. The facilitated association
of ion pairs would be by virtue of a reduction
in the electrical field effect of the membrane.-5
If this depolarization effect is an essentiality,
it would merely be a permissive function for
vascular smooth muscle contraction in that
the depolarization permits the calcium entering from the primary reaction of increased
permeability to penetrate the membrane in
normal quantities to evoke contraction of normal magnitude.
Evidence that the associated ion-pair hypothesis-5 for membrane passage of ions may
be applicable to vascular smooth muscle excitation-contraction reactions is found in the
demonstration that equimolar injections of
the more polarizable calcium iodide produced
greater calcium contractions than did injections of calcium chloride. The finding suggests that the greater contractions from
injected calcium iodide solution were the result of greater calcium diffusion across the
cell membranes in the form of associated ion
pairs of calcium. The finding supports the
speculation made here that transmembranous
movements of calcium during excitation of
vascular smooth muscle are accomplished significantly in the form of associated ion pairs
of calcium, as appears to be the case in skeletal muscle when nitrate or bromide is substituted for extracellular chloride.4'44
In the present investigation, contractility of
vascular smooth muscle was rapidly depressed
and abolished by perfusate inclusion of
EDTA, 10 mM/L, when the EDTA chelated
available calcium ions of the arterial segment.
The apparent discrepancy that some workers
Circulation Research, Volume XI. December 19Qt
937
(e.g., Jang00) but not all (e.g., Medici,51 Bohr
and Goulet10) found, that calcium ion deficiency did not depress contractility of vascular smooth muscle, may be attributed to the
degree of calcium depletion obtained by the
various investigators.
Besides indicating that calcium is essential
for contractility, the following four types of
findings of this investigation are strong evidence that calcium is essentially involved in
coupling membrane excitation to the myoplasmic response of vascular contraction. (1)
The development of arterial muscle contractions arising from elevated potassium perfusion and from epinephrine perfusion was both
immediately inhibited and prevented by simultaneous use of calcium-deionized perfusion
fluid containing EDTA for the chelation of
extracellular or tissue calcium. The rapidity
of this prevention suggests that it was accomplished through removal of extracellular calcium ions and superficial or labile calcium
bound to cell membranes rather than through
removal of intracellular calcium. The EDTA
itself probably did not migrate intracellularly; there is evidence that its distribution
is limited to the extracellular space.52 (2)
Both the sustained potassium contracture and
the epinephrine-induced contractions were
promptly lessened and removed by EDTA
chelation of calcium. (3) Calcium administered simultaneously with the excitatory injection of epinephrine immediately restored the
epinephrine-induced contractions which had
been abolished by EDTA ehelatiou of calcium.
(4) Extracellularly administered calcium ion
in high concentration produced vascular
smooth muscle contractions, and both epinephrine and potassium excitation greatly increased the magnitude of these "direct"
calcium contractions by means of a specific
increase in the cell permeability to calcium.
Thus, the entered calcium which is coupled to
and produced by excitation appears to activate intracellularly the contractile events of
vascular smooth muscle.
The calcium moved into the vascular myoplasm may directly activate the actomyosinATP-ATPase system of vascular smooth
938
WATJOH
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muscle contractility, or the entered calcium
may trigger contraction indirectly by inactivation of a relaxing factor system. Whether
muscle of blood vessels contains a relaxing
factor system inactivated by calcium, similar
to the relaxing factor system of striated muscle,63 remains to be demonstrated.
Injection of excess potassium ion which
caused depolarization and contraction of the
relaxed artery caused, paradoxically, relaxation of the artery already contracted by epinephrine (see fig. 6). Injected magnesium
ions also lessened the epinephrine-induced contractions; this may be attributed to the depressant effect of magnesium on muscle
membrane excitability.64 On the other hand,
potassium ions have an excitatory effect which
is probably intimately related to displacement
of membrane calcium by potassium and potassium reduction of the net internally negative
charge of the membrane (potassium depolarization). The lack of contractile-inducing
action of the injected potassium ions in the
epinephrine-stimulated artery occurred presumably because of pre-existing epinephrine
depolarization of the muscle membranes. The
paradoxical vasodilator effect of the excess
potassium on the epinephrine-constricted artery may have been the result of a lessened
intracellular action of calcium brought about
by increased potassium (and sodium) influx
with increased univalent cationic-calcium competition for anionic sites of the contractile
system itself. Such a potassium-calcium antagonism may underlie both the depressive
part of the dual action of potassium on epinephrine excitation of vascular muscle, which
Bohr and Goulet10 observed, and the relaxation of potassium-poor vascular smooth muscle
as a function of the amount of reaceumulation
of intracellular potassium which Barr observed.41
Summary
A technique of constant-flow perfusion of
isolated segments of dog intestinal arteries
was used to investigate the role of calcium in
contractile excitation of vascular smooth muscle by epinephrine and potassium.
Contractile responses of the arterial muscle
to epinephrine, perfused at constant concentration, and to potassium, perfused at high
concentration, were both promptly prevented
by calcium deionizing accomplished by simultaneous perfusion with solutions containing
ethylenediaminetetraacetate (BDTA) for the
chelation of available calcium ions.
Potassium-induced muscular contracture
was removed in a few minutes and epinephrine-induced contractions of potassium-depolarized muscle were soon prevented by perfusion with solutions containing ionic species of
BDTA which chelated calcium ions. In muscle depolarized by potassium and calcium
deionized by perfusion with solution containing MgKoEDTA, the contractile responses to
epinephrine were immediately restored by
combined perfusate injection of calcium ion
and epinephrine.
Injections of isosmotic calcium chloride solution of subthreshold or threshold contractile
strength, when the artery had been perfused
with sodium chloride—rich solution and the
muscle had been relaxed, produced much
greater contractions during both potassium
and epinephrine excitation. The contractions
from injected calcium chloride solution were
increased by epinephrine excitation of muscle
already depolarized by the external application of potassium sulfate, as they were in the
previously polarized or resting muscle excited
by epinephrine.
The thesis is advanced that the relative
muscular contractions induced by standard
injected volumes of isosmotic calcium chloride
solution were a relative index of the calcium
influx and calcium permeability of the arterial
smooth muscle cells in the various experimental conditions prevailing.
Equimolar injections of isosmotic calcium
iodide solution produced greater muscular
contractions than did injections of isosmotic
calcium chloride solution. The possibility is
discussed that transmembranous passage of
calcium into vascular muscle cells occurs significantly in the form of associated ion pairs.
A dual effect of potassium on vascular
smooth muscle behavior was observed in that
a previously contractile-inducing amount of
Circulation Retearch, Volume XI. December 196!
939
CALCIUM AND VASCULAR SMOOTH MUSCLE
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injected potassium exerted a relaxing effect
on the arterial segment perfused with lowpotassium solution when the muscle was contracted by epinephrine.
It is concluded that: (a) calcium is essentially involved in excitation-contraction coupling of vascular smooth muscle; (b) the
calcium permeability and calcium influx of
vascular smooth muscle is greatly increased
during both epinephrine and potassium excitation of the muscle cells, and epinephrine
accomplishes these calcium changes even in
muscle already depolarized by potassium; (c)
adrenergic neurohormones exert their vasoexcitatory contractile effect by a membrane
reaction which is basically nonelectrical and
which primarily triggers an increased permeability and influx of calcium into the vascular
myoplasm; and (d) the migrated calcium activates intracellularly the myoplasmic events
of vascular smooth muscle contraction.
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Circulation Research, Volume XI. Docombor IBet
Role of Calcium in Contractile Excitation of Vascular Smooth Muscle by Epinephrine and
Potassium
William H. Waugh
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Circ Res. 1962;11:927-940
doi: 10.1161/01.RES.11.6.927
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