membrane is depolarized beyond th

19 Pages·2005·1.63 MB·English

J. Physiol. (1972), 220,pp. 363-381 363
With 14 text-figures
Printed in Great Britain
THE ANTAGONISTIC ACTIONS
OF CALCIUM AND MAGNESIUM ON THE SUPERFUSED
VENTRICLE OF THE SNAIL HELIX POMATIA
BY R. F. BURTON AND J. R. LOUDON
From the Institute ofPhysiology, University of Glasgow, Glasgow, W. 2
(Received 26 July 1971)
SUMMARY
1. The isolated, superfused half-ventricle of the snail (Helix pomatia)
maintains a degree of tonic activity even when not beating, since the
membrane is depolarized beyond the tension threshold. Beating may be
initiated by lateral stretch ofthe ventricle and by 5-hydroxytryptamine.
2. Ca and Mn each have a hyperpolarizing action, while K and to a
lesser extent Na cause depolarization: the tonic activity of the ventricle
is affected accordingly.
3. An increase in the extracellular concentration ofMg also causes the
tension to fall, but without change in membrane potential. It can also
initiate beating.
4. Contractures induced by 30 mm-K are steadily maintained, but start
with a more-or-less distinct twitch-like contraction. A contracture induced
by a concentration of K above about 50 mm is poorly sustained and is
followed by a further brief contracture when the K concentration is
reduced to normal.
5. Relations between contracture tension and the concentrations ofCa
and Mg accord with the hypothesis that the two ions compete for attach-
ment to a binding site on the cell surface and that tension is proportional
to the amount ofbound Ca. On this hypothesis, the apparent dissociation
constant of the Mg complex is 11x4 mm and that of the Ca complex
0x15 mm or less.
6. This effect ofMg is like that ofNa on frog ventricle and some ofthe
differences in the behaviour of snail and frog ventricles are abolished by
appropriate adjustment of the extracellular concentrations ofthese ions.
INTRODUCTION
Muscular contraction is affected by the ions in the extracellular fluid in
two general ways, through their influence on electrical activity and
through their influence on the mechanisms that link electrical activity to
364 R. F. BURTON AND J. R. LOUDON
contraction. Inordertoeliminate the effects ofionsontheactionpotential
of the snail heart and so to learn more of their influence on excitation-
contraction coupling in that tissue, Burton & Mackay (1970) studied
contractures induced by alternating current in the ventricle of Helix
aspersa. Thecontracturetensionintheseexperimentswasfoundtorisetoa
plateau level with increasing concentrations ofCabut to fall withthe con-
centration ofMg. The results suggested a direct competition between the
two ions for binding sites on the cell surface such as has been postulated
for rat uterus (Edman & Schild, 1962) and it seemed desirable to explore
this idea further in relation to KCl contractures in the ventricle of the
larger snail, H.pomatia, andto attempt to assigndissociation constantsto
the complexes between the hypothetical binding site and the two cations.
The data on H. aspersa were unsuitable for the purpose since the non-
beating ventricles had maintained a slight degree oftonus.
Earlier work (Hogben, 1925; Bachrach, 1945; Paul, 1961) has shown
the snailventricleto beinsomerespects like frogventricle initsresponses
to ions and unlike it in others; the two tissues will be compared in this
paper in the light ofthe new observations.
METHODS
Tostudychanges intension, ventricles ofaestivating specimens oftheRoman, or
edible,snail,H.pomatiaL.,weredissectedoutandcutinhalf,longitudinally,insaline.
Theauricularendofahalfventriclewaspinnedflattotheplatformofasuperfusion
apparatus (Lamb & McGuigan, 1966) by means ofthree thin silver pins. The apex
wasattachedbyafinethreadtoatensiontransducer,itselfcoupledtoapenrecorder.
During superfusion, fluids poured onto the preparation, inner surface upwards, at
50-60ml./min. Contracturetensionsweremeasuredastheincreaseabovetheresting
(baseline) tensions.Whentwoormoretensiontraceshavebeensuperimposedinthe
preparation offigures and the instants offluid change have not coincided, only the
firstchangeofagivenkindisshownbyanarrow,theremainderoccurringduringthe
period indicated bythe accompanying bar.
Membrane potentials were recorded with intracellular glass micro-electrodes and
storage oscilloscope. Since superfusion was not then appropriate, the strip of
ventricle was placed in abath instead. Before each series of measurements (eight
totenimpalementsofsuperficial fibres in each solution) the pieceofventricle,after
several washings, was allowed to soak 5min in the new solution.
Two basic salines were used, designated K4-Mgl4 saline and K4-Mg56 saline to
indicate themillimolar concentrations ofK andMg. Bothsolutionscontained: KC1,
4mM; NaCl, 80mm; Tris-(hydroxymethyl)-aminomethane chloride, 8mm; glucose,
1-7mM; pH 7-9. In addition to these, K4-Mgl4 saline contained: MgCl1, 14mM;
CaCl2, 8mM; sucrose, 105mm and K4-Mg56 saline contained: MgC12, 56mM; CaCl2,
1 mM; no sucrose. Other salines, representing modifications ofthese two, are speci-
fied later. The sucrose in K4-Mgl4 saline raised the osmolarity towards the upper
end ofthe natural range (Burton, 1965).
Ca AND Mg ON SNAIL VENTRICLE3365
RESULTS
The flat strip ofventricle did not beat spontaneously when superfused
with K4-Mgl4 saline, but maintained a steady 'base line' tension. How-
ever, when 5-hydroxytryptamine was added in concentrations greater
than 10-8g/ml., the preparation first gave a strong contraction and then
began to beat vigorously and with a regular rhythm. Spontaneous beating
could also be elicited byplacing the three pins close together at the base of
the strip ofventricle and so allowing lateral distension ofits walls by the
pressure of the superfusion. The beat was then irregular and diastolic
relaxation was often incomplete.
a 140
1 g
b 10sec
t2Mn
a
Fig. 1. RelaxationofbaselinetensioninK4-Mgl4salineduetoremovalof
Na or addition ofMn. (a) Superimposed tracings showing the influence of
140, 40and8mM-Na(sucroseassubstitute). ReductioninNaconcentration
from the control of 80mm resulted in a drop in base line tension, while
140mm-Na produced a small rise. (b) Addition of 2mM-Mn to K4-Mgl4
saline produced a reversible drop in base line tension followed by a few
weak beats.
The effects on base line tension ofNa, Mn and Mg
When the sucrose in the K4-Mgl4 saline was replaced by NaCl so that
the concentration of Na was raised from 80 to 140 mm, a small rise in
tension occurred (Fig. 1a) and when the concentration ofNa was lowered
from 80 to 40 mm or to 8 mm, with the substitution of osmotically equi-
valent amounts ofsucrose, theresultwas asmall dropinbaselinetension.
This suggested that the ventricle was not at rest in K4-Mgl4 saline, but
retained some degree oftonic activity.
Asfurtherevidenceofthis,asmalldropintensionoccurred,followedbya
few weak beats, when 2 mm-Mn was added (Fig. 1b).
Another ion which produced a drop in base line tension was Mg. When
its concentration was raised from 14 to 28 mm with the removal of the
osmotically equivalent amount ofsucrose, theresult was afall in base line
tension (Fig. 2b).Further increase to 56 mM-Mg, with Castill at 8 mm, not
366 R. F. BURTON AND J. R. LOUDON
only caused further relaxation but also induced regular spontaneous
beating (Fig. 2c); when K4-Mgl4 saline was re-introduced, the ventricle
halted abruptly in what was clearly a contracture, though this was also
in fact the original base line tension (Fig. 2d). In agreement with these
results, the complete removal ofMg resulted in a rise in tension (Fig. 2a).
This 'Mg-free contracture' was approximately equivalent in tension and
shape to a contracture induced by raising the concentrationofKto30mM
with the concentration ofMg remaining at 14 mM.
a
14Mg t0Mg 1 t30K t
b -IA
14Mg t28Mg 4
14Mg +56Mg
d mu adAd _ 1|Jg
10sec
t14Mg
56Mg +56Mg
Fig. 2. The influence of Mg on base line tension. (a) Mg-free saline pro-
duced awell sustained riseintension, approximately equivalent to that of
the 30mM-K contracture following it. (b) 28mm-Mg produced a small,
reversible, drop in tension. (c) 56mM-Mg produced a drop in tension
followed by regular spontaneous beating. (d) Re-introduction ofK4-Mgl4
saline resulted in an abrupt halt in a contracture which was the previous
base line tension. The effect was reversible.
The K contracture. When the external K concentration was raised to
between 6 and 200 mm by the addition ofKC1 without compensation for
the increased osmotic pressure, a contracture occurred (Fig. 3). The
threshold for this effect was very low, a significant rise in tension being
produced even at 6 mM-K. Lowering K to 2 mm had no effect, but K-free
fluids produced a small contracture.
The form ofthe contracture, as well as the rise in tension, depended on
the concentration of K. Thus, at concentrations below 50 mm, the con-
tracture tension rose rapidly to a well sustained 'plateau' tension. At
higher concentrations, this plateau became increasingly poorly sustained
and was followed by a further contraction when K4-MgL4 saline was re-
introduced. Thereasonforthis 'reboundcontracture'isnot clear.
Ca AND Mg ON SNAIL VENTRICLE 367
Membrane potential
K. The membrane potential in K4-MgL4 saline averaged -59-8 +S.D.
2-7 mV in nineteen ventricles. The dependence ofmembrane potential on
K concentration in the range 0-200 mm was examined with KCl being
added as in the experiments on tension. The concentrations ofother ions
+0K t4K
+6K +4K '
10sec
+10
K 4K
t4
+20K K
50K 4K
t
IF200 K 4K
Fig. 3. The influence ofK on tension. At each arrow the K concentration
in the K4-Mgl4 saline was changed as indicated. 6mM-K produced a
significant rise in tension and the tension increased with furtherrise in K.
Up to 50mM-K, contractures were well sustained, but at higher concen-
trations became less so and were followed by 'rebound' contractures on
re-introduction of K4-Mgl4 saline. K-free fluid also produced a small
contracture.
were as in the standard K4-Mgl4 saline. The results are shown in Fig. 4.
Over much ofthe range, a tenfold increase in K concentration depolarized
the membrane by about 38 mV. Lowering the concentration ofK hyper-
polarized the membrane and the application of K-free saline produced a
hyperpolarization of 9-0+S.D. 2-1 mV (P < 0-001).
Ca. The influence of Ca on the membrane potential was studied by
introducingsalinecontaining 0, 1or16 mm-Cainsteadofthe8 mmnormally
368 R. F. BURTON AND J. R. LOUDON
presentintheK4-Mgl4saline. Theaveragechangesinmembranepotential
withreferencetothoseinK4-Mgl4saline areshowninTable 1;removalof
Ca leads to depolarization and addition to hyperpolarization. When the
concentration ofK was 30 mm, changes in Ca over the range 1-9 mm had
no significant effect on membrane potential.
0
> -25
-
0
01
-oa
EZ-50 _i ~~~~~~~~~/
///T
(0) 1 2 4 10 20 30 50 70100 200
Concentration ofpotassium (mm)
Fig. 4. The influence ofK on membrane potential. Mean values are each
for 3 or 4 ventricles, except that for 4mM-K which is based on 19. Bars
indicate S.D. The interruptedlineis ofgradient 38mY pertenfold change
in K. The influence ofK-free saline is shown to theleft.
TABiE 1. Dependence of membrane potential on [Ca]. Negative sign indicates
depolarization with respect to potential in the presence of8mm-Ca. Other ions are
as in K4-Mgl4 saline
Change±S.D.
m-Ca (mY) Number
0 -8-1+2-3 3
1 _4-9+0-8 5
8 0
16 +3-5±2-1 6
Mg. Complete removal of Mg from K4-Mgl4 saline produced no signi-
ficant change in membrane potential in five experiments. Although higher
concentrations ofMg (28, 42 and 56 mM) in what was otherwise K4-Mgl4
saline caused slow spontaneous beating and made measurement more
369
Ca AND Mg ON SNAIL VENTRICLE
difficult, sufficient impalements were made during diastole to suggest that
the membrane potential was unaltered, atleastthroughmuch of diastole.
Thus the effect ofMg on base line tension andthe initiation ofbeating by
high concentrations seem not to be mediated by changes in resting mem-
brane potential. Mg also appeared to be without effect on membrane
potential in the presence of 30 mM-K. All alterations in Mg were accom-
panied by appropriate changes in the contribution of sucrose to osmotic
pressure.
Mn. The application of 2 mM-Mn, already mentioned as causing a fall
in base line tension, produced, in four experiments, an average hyper-
polarization of 1.8 mV (P < 0.01).
Na. Reducing the concentration of Na in K4-Mgl4 saline from 80 to
8 mm, with replacement by sucrose, produced, in five experiments, an
average hyperpolarization of 7-4 mV (P < 0 01). An intermediate hyper-
polarization (5 mV) resulted from reduction in the concentration of Na
from 80to 40 mMinthreeexperiments, butanincreasefrom 80to 140 mM,
in five experiments, produced no significant change (an average hyper-
polarization of1 0 mV). Inthepresenceof30 mM-K, Nahadnosignificant
effect on membrane potential over a range of 8-80 mm.
Influence ofcations on the contracture induced by 30 mM-K
A K concentration of 30 mm was chosentoelicit contractures, because
the plateau tension is then well sustained (Fig. 3) and so provides a con-
venient measure of contractility. Between contractures, the ventricles
were superfused withK4-Mg56 saline, the high concentration ofMgbeing
intended to minimize the contribution of tonic activity to the base line
tension. In addition the Ca level was loweredfrom 8 to 1 mm to make the
preparations more responsive to change in Mg.
When superfused with this saline, the ventricles initially beat spon-
taneously, as in salines containing high Mg and normal Ca, but stopped
doing so after 10-15 min. An increase in the concentration ofK to 30 mm
(with Ca remaining at 1 mm and Mg at 56 mm) resulted in a contracture
that started with a phasic contraction not unlike a large slow twitch, the
relaxationofwhichwasinterruptedbyasecond, 'plateau',phase(Fig. 5a).
When the concentration of Ca was raised from 1 to 8 mm at the same
time as that of K was raised to 30 mm, the distinction between the two
components was no longer evident. The plateau tension was also greatly
increased despite the short time the raised Ca concentration had in which
to act.
The influence ofMg on the contracture induced by 30 mM-Kwas tested
over the range 0-56 mm, LiCl being used as a substitute to maintain the
osmolarity. Reduction in the concentration ofMg had the same effect on
370 R. F. BURTON AND J. R. LOUDON
the contracture form as an increase in the concentration ofCa, namely a
rise in tension with a merging ofthe two phases of contraction (Fig. 5b).
The contracture in Mg-free 30 mM-K saline was approximately equi-
valent to a contracture in the presence of 8 mM-Ca, 56 mM-Mg and 30
mM-K. These effects of Mg occurred whichever substitution agent was
used; those tested (quoting concentrations used in substitution for 56
mM-MgCl2) were LiCL (80 mM), NaCL (80 mM), choline chloride (80 mm),
Tris-(hydroxymethyl)aminomethanechloride (85mM)andsucrose(145mM).
430K 130K ]1g
4 4
ICa 8Ca
10sec
0 14 28
561Mg 30K4 3I0CKa t3056KMg +
ICa Mg asshown 8Ca
c
4~~t30 L30KL
430K 4 K
56Mg 56 Mg0M~g-Li 0Mg-Choline
-- Na -- Tris
Sucrose
Fig. 5. The influence of Ca and Mg on the 30mM-K contracture. Super-
fusion before and after contracture was always with K4-Mg56 saline.
(a) Two traces ofcontractures, each in the presence of56mM-Mg. During
the firstcontracture Ca remained at 1 mM; the second contracture fluid
(30mm-K) contained 8mM-Ca. (b) TheinfluenceofMg,withCaconstantat
1 mm. The third trace shows,forcomparison, acontractureinthepresence
of 8mm-Ca and 56mM-Mg. (c) The effect ofreplacing MgCl2 with LiCl or
NaCl (second trace) orwithcholinechloride, Tris chloride orsucrose (third
trace).
Complete replacement of Mg by any of these invariably caused an
increase in the tension of the K contracture, although the size of the
effect did varywiththe substitute (Fig. 5c). Thus replacement ofMg with
LiCl or NaCl caused an increase in tension ofabout 100%, while replace-
ment with sucrose or with choline or 'Tris' chloride caused an increase of
about 130 %.
Ca AND Mg ON SNAIL VENTRICLE 371
The dependence of contracture tension on the nature of the substance
replacing Mg suggests that Na and Li each have a negative inotropic
effect, though small compared with that of Mg. Other experiments in-
volving contractures produced by 30 mM-K in the presence of 56 mM-Mg
also suggested this. Thus the contracture tensions were increased by
20-30% whenthe concentration ofNawaslowered from 140to 8 mmwith
the substitution of sucrose, choline chloride or 'Tris' chloride, while no
suchincrease occurredwhenthe osmotic pressure was maintained with Li.
At most, the effect ofNa on contracture tension would seem to be small.
02,
4K 4K 30 K 30 K 4 K
I Ca OCa OCa Caasshown 1 Ca
a b c d e
Fig. 6. This Figure illustrates the sequence of five steps, as described in
the text, involved in studying the influence of Ca on the 30mM-K con-
tracture. This influence is itself illustrated by the superimposed traces
to the right.
The antagonism between Ca and Mg
The quantitative relationships between contracture tension and the
concentrations of Ca and Mg were investigated in experiments in which
the ventricles were exposed to varying concentrations ofthese ions after
first being depolarized by 30 mM-K in the absence of Ca. The procedure
consisted ofthefive steps illustrated in Fig. 6. (a) The ventricle was super-
fused with K4-Mg56 saline containing 1 mM-Ca. (b) Superfusion with a
similar saline, but lacking Ca, then resulted in an immediate drop in
tension. It was from this base line that measurements of contracture were
subsequently made. (c) After 1 to 2 min of superfusion with this Ca-free
K4-Mg56 saline, the latter was replaced by a Ca-free saline containing
30 mM-K and 56 mM-Mg. Usually no tension resulted (as in Fig. 6), and
this was taken to indicate that the ventricle then retained too little Ca to
contract. If any tension did develop, the ventricle was superfused again
inCa-freeK4-Mg56salineforafurther 1-2 min,before the re-introduction
ofthe Ca-free saline containing 30 mm-K. The ventricle was then in a Ca-
free, depolarizedstate, atzero contractile tension. (d) The next stepwasto
372 R. F. BURTON AND J. R. LOUDON
introduce Ca (often with a change in Mg concentration too, as in Fig. 8)
while the concentration of K remained at 30 mm. Contractures then de-
veloped. (e) Finally, there-introductionofK4-Mg56saline causedareturn
to the original tension ofa.
lor
0 0
0
a 0 a
aS A
0
0
0
0
a A
0VI 501-
co
._ 0
2 0
A
Ioa a
.
o UI I
0 4 8
Calcium (mM)
Fig. 7. The relation between plateau tension during 30mM-K contracture
and the concentration ofCa. Tensions are plotted as percentages ofthose
inthe presence of8mM-Ca. The Mgconcentration is 56mm. Each symbol
represents one ventricle.
'--" 14
-.1 56
t
t
4K 30K 30K 30K
OCa OCa ICa 0-3Ca
Mgas shown
Fig. 8. The influence ofMg on the 30mm contracture. A control contrac-
ture is shown in the centre, following depolarization in the Ca-free saline.
The superimposed traces to the right show the influence ofMg (0-56mm)
on contractures induced by 30mn-K in the presence of0-3mm-Ca.
The influence ofadded Ca on the Ca-free depolarized heart muscle was
tested in four experiments. As before, the form and tension of the con-
tracturedependedontheamountofCainthe 30 mM-Ksaline. Thus, atlow
concentrations (0 1-1 0 mM-Ca) the contracture was made up ofaninitial
phasic tension followed by a plateau. Both ofthese componentsincreased
with the concentration ofCa until they merged at about 8 mM-Ca.
TheplateautensionsatthedifferentconcentrationsofCaweremeasured