Published November 1, 1961
Active Ion Transport Across
Canine Blood Vessel Walls
P H I L I P N. SAWYER, JOEL LEVINE, ROGER MAZLEN, and
IGNATIUS VALMONT
From the Department of Surgeryand Surgical Research of the State Universityof New York
Downstate Medical Center, Brooklyn
Applied electrical fields and those created by currents of injury in injured
tissues appear related to intravascular occlusion of blood vessels (1-3).
Abramson, Gorin, and Ponder (4), Furchgott and Ponder (5), Ponder apd
Ponder (6), and others have indicated that the cellular elements of blood at
a p H of 7.40 move toward the positive pole in an electrophoretic cell. Vascular
occlusion can be potentiated in normal vessels by application of a positive
electrode (7, 8). Occlusion is delayed in an injured blood vessel exposed to
the field about a cathode (9, 10). Therefore it is postulated that potential
differences in the physiologic range and related currents of injury in perivascular tissues m a y act as causal agents in intravascular occlusion, by means
of their electrophoretic or other effects upon the cellular and possibly liquid
elements of the contained blood.
As a corollary, it is reasonable to hypothesize that the blood vessel wall
The Journal of General Physiology
Downloaded from on June 15, 2017
ABSTRACT Experiments giving evidence of active Na and C1 ion fluxes
across large canine blood vessel walls (aorta, vena cava) in vitro have been
presented. The information has been obtained using ion tracer techniques after
Ussing and with diffusion cells of the Hogben type. The available data suggest
that the membranes are satisfactorily oxygenated by the bathing solutions
saturated with oxygen at atmospheric pressure. Evidence is offered which
indicates that active ion transport does occur across the aorta and vena cava
in in vitro experiments. Under the conditions of the experiment net Na and C1
flux takes place from intima to adventitia across the aorta, and from adventitia
to intima across the vena cava at low measured potential differences. The
possible relationships of derangement of active ion transport mechanisms,
produced by electric currents and tissue injury potential differences, to intravascular thrombosis are alluded to. It would appear that sodium and chloride
fluxes across large blood vessel walls in vitro occur at least in part as the result of
metabolic processes and cannot be explained simply on the basis of diffusion
across a semipermeable membrane.
Published November 1, 1961
i82
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 45 " 196I
ordinarily performs work in order to neutralize transmural potential differences and adventitious currents occurring in the living organism possibly
by the active transport of ions. If this were not possible, minor injuries could
result in a chain of events causing progressive, unrelenting intravascular
occlusion and widespread ischemia with necrosis. Thus, it seemed reasonable
to postulate that one function of the blood vessel wall consists of active transport of ions to control transvascular ion concentration and electric charge.
T h e experiments described in this paper are the first of a series designed to
test this possibility.
EXPERIMENTAL
TECHNIQUE~
MATERIALS,
AND
METHODS
Experimental Membranes
TABLE I
KREBS SALINE S E R U M S U B S T I T U T E
Mix:
8O parts 0.90 per cent NaCI (0.154 M)
4 parts 1.15 per cent KC1 (0.154 M)
3 parts 0.11 M CaC12
1 parts 2.11 per cent KH2PO4 (0.154 M)
1 parts 3.82 per cent MgSO4.7 H~O
21 parts 1.3 per cent NaI-ICO~, (0.154 M); treated with GO~ until p H is 7.4
f4 parts 0.16 • Na-pyruvate (or L-lactate)
17 parts 0.I M Na-fumerate
parts 0.16 ~ Na-L-glutamate
parts 0.3 M 5.5 per cent glucose
* Prepared by neutralizing a solution of the acids with M NaHCOa solution.
TABLE II
B L O O D VESSEL W A L L P R E P A R A T I O N S
A. Aorta
1. Fresh
2. One day storage at 4°C.
B. Separated aortic wall layers
1. I n t i m a (inner layer preparation)*
/ •
.
,~rMatched specimens from same aorta
2. Adventitia touter layer preparation)~}
3. Intimal p r e p a r a t i o n alone
C. V e n a cava
D. Control membranes
1. Dialysis m e m b r a n e
2. P a r c h m e n t p a p e r
* Henceforth called intima for sake of brevity.
:~ Henceforth called adventitia.
Downloaded from on June 15, 2017
It was necessary to measure ion fluxes across blood vessel walls in vitro in order to
obtain accurate measurements. The dog blood vessel wall was used as the experi-
Published November 1, 1961
SAWYER, LEVINE,MAZLEN, AND VALMONT Ion Transport in Blood Vessel Walls
183
mental m e m b r a n e because it was easily accessible and because oxygen utilization
studies had already been performed on the canine aorta by Kirk, Effersoe, and
Chiang (10). T h e y report that canine and h u m a n aortae consume oxygen for several
weeks after being removed from the experimental animal, and give rates for oxygen
uptake by the aortic wall.
Vascular membranes for this experiment were obtained from mongrel dogs
sacrificed by bleeding, intravenous pentothal, or air embolism. T h e membranes
included canine descending thoracic aorta and thoracic superior and inferior vena
cava which were removed within 3 to 4 minutes after death. T h e vessel was placed
immediately in Krebs' saline serum substitute (11) (Table I), and either stored at
4°C, or used immediately. Aorta was incised longitudinally between the paired
intercostal vessels. Prepared membranes were placed between ion flux chambers.
Most experiments were run in quadruplicate. I n the first ninety-seven experiments,
the entire thickness of aortic wall was used. In the next series of fifty experiments the
intima and subintimal layers were separated from the muscularis and adventitia in
the e n d a r t e r e c t o m y plane (Fig. 1). T h e inner layers (intima and elastica interna,
Table I I ) alone, or m a t c h i n g separated inner and outer layers of the vessel were used
for certain of the differential ion flux experiments. Experiments were also carried out
using the vena cava wall as the experimental m e m b r a n e (Table II).
Downloaded from on June 15, 2017
FIGURE l. Illustration of a typical segment of aortic wall. The three classical histological layers, intima, media, and adventitia, are indicated, as is the single layer of
endothelium. Note that the vasa vasorum and capillaries which perfuse the arterial wall
end at the inner third of media where they form a capillary plexus. Most histological
studies indicate that the inner wall of the aorta and most blood vessels are not normally
penetrated by the vasa vasorum (23).
Published November 1, 1961
I84
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
•
VOLUME
45
"
I96I
Experimental Technique
Each of four specimens from the same blood vessel was placed between diffusion cell
pairs of the H o g b e n type (12, 13) (Fig. 2). T e n ml of K r e b s ' serum saline substitute
at p H 7.40 a n d a t e m p e r a t u r e of 37°C (11), was placed in each of the cells to bathe
each side of the specimen. T h e solutions were oxygenated with 95 per cent O2-5 per
cent COs which b u b b l e d in at the b o t t o m of the cell. Each m e m b r a n e h a d a n exposed surface area of 1.0 sq. cm.
After 15 to 20 m i n u t e s for equilibration, the radioactive isotopes were added to
the d o n o r solution. Experiments r u n for as long as 10 hours showed evidence of water
e v a p o r a t i o n with ion concentration. These findings resulted in our using a d u r a t i o n
of 4 hours for a n experiment.
Radioisotope Measuring Techniques
S o d i u m m o v e m e n t across the aortic wall was first d e t e r m i n e d using 1 microcurie of
N a ~2 in the d o n o r solution. T h e initial experiments were done c u m u l a t i v e l y so that
if n e t u n i d i r e c t i o n a l transport occurred it would be d e m o n s t r a b l e after 2 to 3 hours
Downloaded from on June 15, 2017
FIGURE 2. Photograph of cells used to measure ion flux across blood vessel wall. The
cells separate to allow insertion of membranized blood vessel wall into the 1 cm ~ opening
between the two chambers. They are then clamped shut, the needle oxygen bubblers
inserted in the bottom, the ceils sealed with melted paraffin, and the bathing solution
added. Current electrodes are inserted in the ends of the cells; P.n. measuring electrode
bridges inserted from above. Mixing is carried out by the gas jets from below. Proof of
adequate mixing has been obtained using parchment paper and dialysis membrane as
control membranes.
Published November 1, 1961
SAWYER., LEVINE, MAZLEN, AND VALMONT Ion Transport in Blood Vessel Walls
185
Counting Techniques
Counts of the beta emission from both Na 24 and CP 8 were taken using a Baird atomic
model 132 scaler and a shielded gas flow Geiger-Muller counter. Duplicate counts
have been found accurate to 2 per cent. The determination of Na 22counts was carried
out by a single count. Initial and repeat determinations for differential Na 24, C138
counts were made several days apart. After an initial count, immediately after drying,
which effectively measured the emission for both isotopes, the Na 24 emission was
allowed to decay through ten to eleven half-lives (6 days to I week) leaving only
CP 6 as the material to be counted. The initial Na 2. determinations were corrected for
decay during the experiment because of its short half-life (15.2 hours).
Flux was calculated in micromoles, per square centimeter of membrane, hour of
experiment. Mean hourly fluxes for each experiment were calculated. The duplicate
mean fluxes in each direction were determined, and net flux determined by subtracting efflux from influx. A " T " test for significance of the experimental data in
each group of experiments was calculated.
The sign test was occasionally used to obtain a quick comparison of experiments in
any given group.
In an attempt to check the magnitude of absorption of radioactive isotope by the
membrane itself, as a source of discrepancy in the determinations, the experimental
membranes were themselves counted at the end of 50 experiments. At no time did
radioisotope concentrations within the membrane at the end of a 4 hour experiment,
exceed 0.1 to 0.5 per cent of the total isotope placed in the solution on the donor side
of the cell (14). Does the isotopic loss caused by hourly removal of the isotope in the
Downloaded from on June 15, 2017
because of progressive separation of the curves of Na flux in opposite directions, even
though progressive cell asymmetry would result in increasing back diffusion as net
transport progressed in one direction. In subsequent experiments 1 microcurie of
Na ~2 or Na 24 and 1 microcurie of C186each diluted with 0.9 per cent NaC1 were used
to determine non-cumulative differential fluxes of the two ions across the same
segment of blood vessel wall.
Na 24 is received as Na~4COa: 5 to 10 m g / 1 0 millicuries. This was diluted by large
amounts of 0.9 per cent NaC1 in each instance so that the isotope was added to the
bathing solution essentially as physiologic saline solution. 250 microcuries of C138 are
received as 2 cc of dilute hydrochloric acid. After neutralization with sodium bicarbonate, the C1s6 was further diluted with 250 cc of 0.9 per cent NaC1 to make 1 #c
C136 per cc.
One microcurie of each of the isotopes was placed in the bathing solution on the
intimal side of the membrane in two of the cells and on the adventitial side of the
membrane in the remaining two pairs of cells. After mixing, 1 cc of donor solution
was removed from each cell, dried, and counted, yielding a master donor count for
calculation of the per cent of isotope which traversed the membrane per hour.
At the end of each hour after the experiment had begun, the entire recipient
solution was removed from the flux cells to limit the amount of back diffusion. One
cc samples of recipient solution were dried in aluminum planchets and counted.
Published November 1, 1961
I86
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
45 " 196I
recipient solution appear to affect the experiment? In the aortic experiments less
than 2 to 5 per cent of total isotope traversed the membrane per hour from the donor
to the recipient solution. At the end of 4 hours, this amounted to 8 to 20 per cent of
the total isotopic and non-isotopic Na and C1 in the donor solution.
In the vena caval experiment net fluxes were five to ten times those of the aortic
experiment and this possibility appears more possible. However, the Na and C1 flux
curves flatten out following the 1st hour and it would appear that the decrease in
available donor ion does not result in an appreciable decrease in flux. Thus the.Na
and C1 flux remains fairly constant once the isotopes of Na and C1 reach equilibrium
across the membrane.
The possibility of Na or C1 leak out of the blood vessel wall membrane in sufficient
quantity to cause an effective change in Na and C1 concentrations in the bathing
TABLE
III
Beginning
Aorta
Vena cava
87.1
No direct data
available
Average wet net weight,
I eros of canine blood
After 4 hrs.
vessel wall (Sawyer)
115.0
Total Na content for
canine blood vessel
Beginning
After 4 hrs.
0.2045gm (16)*
0.018 m.eq
0.023 m.eq
0.145 gm (16)*
0.0126:~
0.0166~
* Figure in parantheses is number of determinations
Assuming that vena cava has approximately the same chemical composition per unit weight
as aorta.
From Bevan (15).
solutions must be considered. In a recent article Bevan (15) (Table I I I ) has indicated
that rabbit aorta has an Na concentration of 87.1 m.eq/kg, which after bathing 4
hours in Krebs' saline solution increased to 115.0 4- 3.7 m.eq/kg. Based on these
figures and assuming that dog and rabbit aortae do not contain grossly different
amounts of Na, the total Na content within the exposed aortic membrane (1 cm *) is
17.8 to 23.5/~ e q / c m ~ of exposed membrane and for the vena cava if the membranes
are comparable, 12.6 to 16.6/~ e q / c m 2 (Table I I I ) . In addition the available information indicates that Na + and by inference C1- increases rather than decreases within
the membrane during an experiment. Since each side of the diffusion cell contains
1694 to 1848/~moles of NaCI (depending on whether 11 or 12 cc of Krebs' solution
are placed in the cells), even total unidirectional extravasation of non-isotopic NaC1
from membrane into the bathing solution would change isotope concentration less
than 1 per cent.
During experiments duplicate isotope flux determinations in each direction, intima
to adventitia, and adventitia to intima, were obtained. Gross discrepancies between
the duplicate determinations were considered as an indication of poor mixing, cell
leak, or defect in the membrane, and these required that the experiment be discarded.
Downloaded from on June 15, 2017
Concentration of Na in rabbit
blood vessel bathed in
Krebs saline, m.eq/kg
Published November 1, 1961
SAWYER, LEVlNE, MAZLEN, AND VALMONT Ion Transport in Blood Vessel Walls
187
Control flux experiments were performed using a 5 m/z/pore dialysis semipermeable
m e m b r a n e between the cells.
At the beginning and end of the first fifty experiments p H ' s were measured to
ascertain the extent of change during the course of the experiments. The largest p H
changes found during the course of an entire experiment were in the range of 0.1
p H unit.
Electrical Measurements
Experimental Variables
In an effort to determine the physical chemical characteristics of blood vessel walls
with respect to ion fluxes across them, several experimental variables were introduced. These included experiments to determine the effects of:
A. Using vena cava as well as aorta for the flux determinations (Table II).
B. Aerobic and anaerobic conditions on matched pairs of specimens from the
same aorta, or the effects of intermittent aerobic and anaerobic periods on
ion fluxes across the same specimens of aorta and vena cava. Duplicate
experiments were not run in this group of experiments. Two cells were run
under aerobic conditions, two under anaerobic conditions.
C. Varying temperatures on ion flux rates and direction (16).
D. Separating the blood vessel wall into layers to determine which, if any, are
predominantly responsible for ion fluxes. The experiments in this group have
been limited to the aortic wall.
RESULTS
T h e c o m b i n e d results for several g r o u p s of studies c o m p l e t e d d u r i n g the
first t h r e e h u n d r e d p a i r e d flux e x p e r i m e n t s using a o r t i c a n d v e n a c a v a l wall
Downloaded from on June 15, 2017
Electrode bridges containing Krebs' agar were placed in close approximation on
each side of the m e m b r a n e and connected via two calomel half-cells to a high impedance recording potentiometer for determination of transmembrane potentials.
T h e spontaneous, electrical potential of the aortic wall in this arrangement was
measured on approximately one hundred occasions. The measured asymmetry of
the circuit (liquid junction asymmetry due to the fluid interface and calomel cell
asymmetry) at the bridge junction was at most 0.1 m v in any given experiment after
calibration. This asymmetry was measured by placing the bridge tips in a common
Krebs' solution pool. The potentiometer was calibrated daily to 0.05 m y against a
standard calibrator accurate to 0.01 my. The stated r a n d o m instrumental error of
the Leeds and Northrup recorder is 1 per cent of the full scale reading. T h e potentiometer was set at zero before and after each measurement of potential difference. T h e
electrode asymmetry was monitored continuously in instances when potential differences were recorded for an entire experiment, since spontaneous m e m b r a n e potential
differences were small.
Published November 1, 1961
i88
THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
45
" 196I
are shown in Table IV. No statistical evidence of net transport was found in
the control experiments using dialysis m e m b r a n e as the control m e m b r a n e
(Table IV, row 8).
Net transport of both sodium and chloride ions occurs from intima to
adventitia across the aortic wall in vitro under the conditions of this experiment
(Table IV, row 1).
TABLE
IV
Net Na flux
Experimental conditions
No. of
experiments*
Direction
Flux~
magnitude
Net CI flux
P value
No. of
experiments
Direction
Flux:~
magnitude P value
Gas
1. F r e s h
aorta
2. E x t e r n a l
aorta
1 day
3. A o r t a
4. A o r t a
intima
5. A o r t a
intima
6. A o r t i c
adventitia
7. V e n a
cava
8. D i a l y s i s
membrane
control
02
15
1 --* A
1.4
0.004
10
1 --~ A
02
8
1 ---* A
1.3
0.03
--
--
N~
02
6
15
A --~ I
A ~ I
3.9
9.8
0.001
0.001
.
Nz
6
None
--
0.35
.
.
.
.
O2
8
None
--
0.21
.
.
.
.
02
8
A --* I
15.5
0.02
8
02
11
--
0.98
11
~ / o n 2 , hr.
None
t~/~,
.
6
.
A ---* 1
A --~ 1
hr.
1.25
0.015
.
15.8
0.005
18.0
0.013
None
--
0.50
* All membrane preparations from one blood vessel are considered one experiment.
:~ M e a n h o u r l y f l u x f r o m f o u r h o u r l y d e t e r m i n a t i o n s .
D o m i n a n t sodium ion flux across the 1 day old canine aortic wall stored
at 4°C is also from intima to adventitia and of similar magnitude (Table IV,
row 2). Experiments to determine chloride flux across stored aortic wall
have not been carried out.
Results with paired aerobic and anaerobic specimens from the same aorta
indicate that anaerobic conditions cause reversal of net sodium flux (Fig. 3),
(Table IV, row 3), (16). Under anaerobic conditions net sodium flux occurs
from adventitia to intima (Table IV, row 3).
Aortic Inner Layer and Outer Layer Experiments
In an additional series of experiments, matched pairs of separated inner and
outer layers of the aorta were run in an effort to determine, which if any,
layer was predominantly responsible for net sodium and chloride flux. In
Downloaded from on June 15, 2017
Vessel
Published November 1, 1961
SAWYER, LEVINE, MAZLEN, AND VALMONT Ion Transport in Blood Vessel Walls
I89
a n o t h e r series the i n n e r l a y e r p r e p a r a t i o n alone was used. O n e notes t h a t
net t r a n s p o r t for b o t h s o d i u m a n d chloride across the i n n e r (intimal a n d
subintimal) layers occurs f r o m the adventitial to i n t i m a l side, just the op-
15
N2 ( A ~ I )
~ s SJ
/
E
0
I0
/
. . _ - - _- - - - - _ -
-~'' ~
o ~"
~
°
0 2 (I--)--A)
NZ I I ~ A
~
o
~
'
02 (A--~T)
x
_J
LL
0
/
Z
0
•
I
[
I
I
I
2
3
4
TIME (hrs.)
F I o u ~ 3. Plot of mean hourly sodium flux across the aortic wall of fourteen pairs of
matched aerobic, anaerobic experiments. Note the reversal of net aerobic Na flux when
anaerobic conditions are imposed. Net flux is also greater under anaerobic conditions.
This increase has not been explained.
A ~ I, indicates flux from adventitia to intlma (influx).
I --~ A, indicates flux from intima to adventitia (efflux).
N2 --* , indicates perfnsion of cells with 95 per cent N2-5 per cent CO2.
O~ --* , indicates perfusion of cells with 95 per cent 02-5 per cent CO2.
posite of t h a t w h i c h occurs across the i n t a c t a o r t i c wall. T h e r e is n o net flux
o n either a n i n d i v i d u a l e x p e r i m e n t a l or o n a statistical basis across the o u t e r
(medial a n d adventitial) layers of the aortic wall in these e x p e r i m e n t s ( T a b l e
I V , rows 4 a n d 6), (Fig. 4).
I f the e x p e r i m e n t is c a r r i e d o u t u n d e r a n a e r o b i c conditions, the aortic
Downloaded from on June 15, 2017
/
/
/ /
iS
Published November 1, 1961
I9o
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
45
• VOLUME
"
196I
i n t i m a displays no statistical e v i d e n c e of net s o d i u m flux in the first small
series of six e x p e r i m e n t s ( T a b l e I V , r o w 5). D e t e r m i n a t i o n s for chloride ion
h a v e not yet b e e n c o m p l e t e d .
57
54
51
~"--"
---- "---
A
>'1"
48
~
45
~
AORTIC INNER LAYER
PREPARATION
42
¢-
39
04
E
36
33
=,
30
x
27
._1
LL
24
o
21
z
~" "-- ~"" .
.I"1-I
18
.
.
.
A
.....
15
~'~
,
AORTIC OUTER LAYER
PREPARATION
12
9
6-
3C
I
I
I
2
TIME
I
3
I
4
(hrs.)
FmuP.~ 4. Comparison of net Na flux across matched inner and outer layer aortic
preparations. These studies indicate that the inner histologically intact aortic layers are
predominantly responsible for net Na + and probably CI- flux.
rgna Cav(l
N e t t r a n s p o r t of b o t h s o d i u m a n d chloride across the v e n a c a v a occurs f r o m
a d v e n t i t i a to i n t i m a ( T a b l e I V , r o w 7). T h e m a g n i t u d e of this n e t flux is
a p p r o x i m a t e l y five to ten times t h a t of the aortic flux w i t h r e s p e c t to b o t h
Downloaded from on June 15, 2017
(.)
Published November 1, 1961
SAWYER, LEVm'E,MAZLEN, AND VALMONT [on Transport in Blood Vessel Walls
,9 t
ions, a n d takes place, in the opposite d i r e c t i o n ( T a b l e I V : c o m p a r e aortic
m a g n i t u d e a n d direction, rows 1-2, with v e n a caval m a g n i t u d e a n d direction,
r o w 7).
O n e also notes t h a t the net flux across the i n n e r layers of the aortic wall is
similarly f r o m a d v e n t i t i a to i n t i m a with a m a g n i t u d e a p p r o x i m a t e l y five
times t h a t for the i n t a c t aortic wall. T h u s , u n d e r identical conditions the
i n n e r aortic layers seem to parallel the v e n a c a v a b o t h with respect to direction a n d m a g n i t u d e of net ion t r a n s p o r t of b o t h s o d i u m a n d Chloride ions.
T h e voltage d e t e r m i n a t i o n s indicate t h a t the s p o n t a n e o u s in vitro P.D. of
a o r t i c a n d v e n a caval wall is v e r y low, of the o r d e r of 1 m v for a o r t a a n d
e v e n smaller for v e n a cava. T h e d a t a indicate, as originally d e m o n s t r a t e d
in vivo (14), t h a t the i n t i m a is slightly negative to the a d v e n t i t i a u n d e r the
TABLE
V
Mean P.D.
Aorta
Vena
cava
No. of
determinations
T test for
Probabilitythat numberis not signifi-
significance
candy different from zero
0.995
42
1.879
0.032 (3.2 per cent)
0.019
19
0.457
0.030 (30 per cent)
Aorta
Pt/Hg/Hg~C12/3 ~ KCl/Krebs/Intima-*Media/Krebs/3 M KCI/Hg2CI2/Hg/Pt
conditions of the e x p e r i m e n t ; i.e., with the vessel b a t h e d with identical solutions o n e a c h side. T h e s p o n t a n e o u s m e a s u r e d aortic t r a n s m u r a l P.D.'S h a v e
r a n g e d f r o m 8 to 0.9 m v with a m e a n P.D. = 0.99 mv. V e n a caval P.D.'S
a v e r a g e m u c h closer to a n d not statistically different f r o m zero with a m e a n
c a l c u l a t e d P.D. = 0.019 m v ( T a b l e V).
DISCUSSION
T h e s e e x p e r i m e n t s indicate t h a t active t r a n s p o r t of N a a n d Cl ion does o c c u r
across large c a n i n e blood vessel walls. W h e n statistical analysis of results of
e v e r y e x p e r i m e n t is m a d e , a net flux of s o d i u m a n d chloride is f o u n d for e a c h
of the vessel m e m b r a n e s studied. This flux o c c u r s in the virtual absence ol an
electrical gradient.
F o r a n y u n i v a l e n t ion w h e n ion m o v e m e n t across the m e m b r a n e is entirely
passive:
/~
Ci
L-coz
Downloaded from on June 15, 2017
MEASUREMENT
OF SPONTANEOUS
P.D. U N D E R
C O N D I T I O N S O F IN V I T R O E X P E R I M E N T
Published November 1, 1961
THE
~9 2
where
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
45
'
I961
f~
fo
C~
Co
=
=
=
=
flux in
flux out
concentration of ion in question bathing the inside of the wall
Concentration of ion bathing the outside of the wall
Z
=
e BvlRT
C~ = Co in these experiments.
At 37°C the P.D. in millivolts = 61 l o g ~ ,
if ion m o v e m e n t across the
m e m b r a n e is passive (17).
For both the sodium and the chloride ions, the ratios of outflux to influx
differ in the three preparations, the aorta, the aortic inner layer, and the
TABLE
VI
Sodium
Mean
measured
P.D.
P value
for P.D.*
M e a n P.D. calculated
from flux ratios
my
Mean
measured
P.D.
P value
for P.D.*
mo
Aorta
--43.1 (39):~
1.0"
0.0001
--55.0 (33)
1.0
0.001
Vena cava
--74.05 (16)
0.01"
0.001
--51.38(20)
0.01
0.0055
* Possibility that the calculated P.V. values, based on Ussing's equation, and the
~.v.'s for aorta and vena cava are not significantly different.
Numbers in parenthesis are the number of determinations.
measured
vena cava, from ratios predicted on the basis of passive diffusion and the
measured potential differences (Table VI). Therefore, according to the definition of Rosenberg (18), Na and C1 must be crossing the m e m b r a n e by active
transport.
It is possible that other ions such as K, Ca, P O 4, and M g are being transported by the vessel wall. No experiments to measure fluxes of these ions in
opposite directions have yet been carried out. If active transport is going on,
either ions with like charge must be transported in opposite directions, or
those of opposite charge in the same direction to keep transmural P.D.'S at a
low level. Since Na and C1 most frequently appear to be transported in the
same direction, their charges would tend to cancel each other.
Experimental evidence that the measured net flux is not an artifact due to
m e m b r a n e asymmetry or non-mixing, is obtained from various elements of
the experiments. Measured sodium and chloride net fluxes are shown to occur
in opposite directions when intact aortic wall is c o m p a r e d to either vena
caval wall or the aortic inner layer preparation; the m a g n i t u d e of net flux is
greater for both inner aortic wall preparations and vena cava than could be
Downloaded from on June 15, 2017
Mean P.D. calculated
from flux ratios
Chloride
Published November 1, 1961
SAWYER, L~.VNE, MAZLEN, AND VALMONT Ion Transport in Blood Vessel Walls
i93
explained on the basis of differences in thickness between these membranes
and the intact aortic wall. Net transport across the aortic wall occurs in
opposite directions in matched aerobic and anaerobic experiments. It would
appear improbable that the various differences listed would occur consistently
if the membranes studied were only passively permeable.
Calculations have been m a d e concerning the possibility that the aortic
wall might be too thick for satisfactory oxygenation and that the observed
phenomenon of reversal of flux occurs for this reason.
TABLE
Wall thickness
Max
¢m
Min
6m
VII
Qog Kirk
Mean
et al. ( z o )
Min.
Max.
0.030
0.62
Max. permissible thickness, cm~:
Warburg (r9), Stevenson (2o), Hill (~ : )
¢m
0.20
0.06
0.10 (20*)
V e n a cava
0.038
0.025
0.030 (14")
Qo~ = 0.03
1.15 cm
Qo2 = 0.62
0.245
Lee, Yu, Sawyer (22)
0.49
0.59
Qo2 = 0.49
o.29
Qo2 = 0.59
0.26
* No of determinations.
:~M a x i m u m permissible thickness in centimeters as calculated from :
W a r b u r g ' s (19) equation:
a, = , , / 8 co x D/A
Where d' = limiting thickness of the membrane.
Co = oxygen tension in atmospheres.
D = 1.64 X 10-5 cc of O2/min. through 1 cm 2 of tissue with a pressure gradient of 1
a t m / c m at 37°C. Value from K r o g h ' s original calculation.
Qo, X 1000
A =
Stevenson and Smith (20)
1000 X 60 X wet w e i g h t / d r y weight ratio
Wet weight
ratio for canine and human aorta = 5.00 (26).
Dry weight
Equations from Warburg (19), Stevenson (20), and Hill (21) indicate that
the aortic wall of m a x i m u m thickness equal to 0.245 to 1.15 cm is thin enough
to be oxygenated satisfactorily by a bathing solution on each side (saturated
with 02 at one atmosphere pressure) at the Qo2 for canine aortic wall determined by Kirk et al. (10) and for canine vena cava by Lee, Yu, and Sawyer
(22), Table VII.
Since net Na flux across the aorta is not abolished but reversed on switching
from aerobic to anaerobic conditions, we must postulate at least two opposing
active transport mechanisms. Studies by Kirk et al. (10) show that approximately 60 per cent of the energy produced by aerobic metabolism is produced
by anaerobic glycolysis, indicating that energy for reversal of active transport
is available under anaerobic conditions.
Downloaded from on June 15, 2017
Aorta
Published November 1, 1961
194
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
•
VOLUME
45
• 196I
Downloaded from on June 15, 2017
Paradoxically net Na and C1 aortic flux occurs in opposite directions across
the intact aortic wall when compared to the aortic inner layer preparation.
T w o possible explanations for reversal of transport can be postulated. O n e
of these would indicate that since no active transport of N a or C1 takes place
across the media and adventitia of the aortic wall in these experiments
(Table IV, row 6), these layers act as a limiting factor in ion influx, since
ions must diffuse through the aortic media to get to the intima and become
available for transport.
T h e available evidence indicates that the aortic inner layers act as the
effective flux membrane. Blood vessels appear to penetrate the outer layers
of the arterial wall in vivo to the level of the inner media where they form a
capillary plexus (Fig. 1). It would appear probable that ion transport in vivo
need only occur across the intact inner layer m e m b r a n e of the arterial wall,
the necessary ions being transported to and from these layers through the
vasa vasorum and capillaries (23). This perfusion source is lost when the
vessel is removed from the experimental animal. W h e n one separates the
inner layer from the remainder of the aortic wall, the amount of sodium and
chloride which can diffuse to the inner intimal cellular m e m b r a n e through
the adventitial side of the inner layer preparation probably approximates
more closely that which occurs in vivo. Ion flux rates for the aortic inner
layer preparation approach ion flux rates across the vena cava. This explanation implies that Na and C1 flux in vivo takes place from adventitia to intima.
A second possible explanation for aortic sodium and chloride ion effiux
when compared to the aortic inner layer preparation influx suggests that u p o n
separating the layers, considerable injury is produced in the aortic wall. T h e
injury so produced m a y result in reversal of the net transport. It has not yet
been possible to determine which if either of these two arguments is more
valid. Additional studies are being carried out.
Do these experiments in any w a y relate to normal ion transport across the
blood vessel wall in vivo? The experiments are carried out under conditions
which do not approximate those of the vessel in vivo. It would be helpful if
the results could be compared to those of in vivo experiments carried out to
study the same problem. Evidence from several investigators indicates that
the cerebral capillary wall does not obey simple D o n n a n equilibrium requirements (24, 25). This m a y be an indication that the phenomena herein
described occur in vivo. Whether or not transvascular active transport is
actually related to vascular homeostasis and the prevention of intravascular
thrombosis due to injury currents or injury potentials in surrounding tissue,
or other resultant thrombotic mechanisms has still to be determined.
T h e phenomena described obviously relate to ion movement between the
intravascular-extravascular space. If transport occurs in vivo as it does in
vitro, it can be postulated that ion effiux on the arterial side of the vascular
Published November 1, 1961
SAWYER, LEVINE,MAZLEN,AND VALMONT Ion Transport in Blood Vessel Walls
195
tree and ion influx on the venous side of the vascular tree are at least partially
due to active transport.
The authors wish to give thanks to Dr. C. A. M. Hogben, Dr. K. S. Cole, and Dr. E. E. Suckling
for their help and thoughtful criticism and continuing suggestions during the course of these studies,
and to Mr. Peter Nemenyi for help with the statistical evaluation of the data.
This work was assisted in part by grants-in-aid from the National Institutes of Health, National
Heart Institute (H-3879), Bethesda, Maryland, and the American Heart Association.
Dr. Sawyeris a Markle Scholar in Medical Science.
Receivedfor publication, October 12, 1960.
BIBLIOGRAPHY
Downloaded from on June 15, 2017
1. SAWYER, P. N., SUCKLING, E. E., and WESOLOWSKI, S. A., The effect of small
electric currents on intravascular thrombosis in the visualized rat mesentery,
Am. 3". Physiol., 1960, 198, 5.
2. SAWYER,P. N., and PATE,J. W., Electric potential differences across the normal
aorta and aortic grafts in dogs, Am. 3". Physiol., 1953, 175p 113.
3. SAWYER,P. N., and WESOLOWSKI, S.A., The current of injury and intravascular
thrombosis, Ann. Surg., 1961, 153, 34.
4. ABRAMSON, H. A., GORIN, M. H., and PONDER, E., Electrophoresis and the
chemistry of cell surfaces, Cold Spring Harbor Syrup. Quant. Biol., 1940, 8, 72.
5. FURCHOOTT,R. F., and PONDER, E., Electrophoretic studies on human red blood
cells, or. Gen. Physiol., 1941, 24, 4.
6. PONDER, E., and PONDER, R. V., Electrophoretic mobility of red cells and their
ghosts as observed with iml~roved apparatus, J. Exp. Biol., 1956, 32, 175.
7. SAWYER,P. N., and WESOLOWSKI,S. A., Experiments in direct current electrocoagulation, Surgical Forum of the American College of Surgeons, Chicago,
American College of Surgeons, 1960, 10, 435.
8. SAWYER,P. N., and PATE,J. W., Bioelectric phenomena as an etiologic factor in
intravascular thrombosis, Am. or. Physiol., 1953, 175, 103.
9. SAWYER, P. N., and DEUTCH, B., The experimental use of oriented electrical
fields to delay and prevent intravascular thrombosis, Surgical Forum of the
American College of Surgeons, Philadelphia, W. B. Saunders Company,
1954-55, 5, 173.
I0. KIRK, J. E., EFFERSOE, P. G., and CmANO, S. E., The rate of respiration and
glycolysis by human and dog aortic tissue, or. Gerontol., 1954, 9, 10.
11. KREBS, H. A. Body size and tissue respiration, Biochim. et Biophysica Acta, 1950,
49 249.
12. HOOEEN, C. A. M., Active transport of chloride by isolated frog gastric epithelium, Am. J. Physiol., 1955, 180, 641.
13. HOOBEN, C. A. M., personal communications.
14. SAWYER, P. N., and VALMONT,I., unpublished data.
15. BEVAN,J. A., The use of the rabbit aortic strip in the analysis of the mode of
action of L-epinephrine on vascular smooth muscle, J. Pharmacol. and Exp.
Therap., 1960, 129,417.
Published November 1, 1961
I96
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
45
• I961
30, 9.
Downloaded from on June 15, 2017
16. SA~WYER,P. N., and VALMONT,I., Evidence of active ion transport across large
canine blood vessel walls, Nature, 1961, 189, 470.
17. USSING, H. H., The active ion transport through the isolated frog skin in the
light of tracer studies, Acta Physiol. Scand., 1949, 17, 1.
18. ROSENBERG,T., The concept and definition of active transport, Syrup. Soc. Exp.
Biol., 1954, 8, 27.
19. WARBURG,O., Versuche an iiberlebendem Carcinomgewebe, Biochem. Z., 1923,
142, 317.
20. STEVENSON, ][. P., and SMITH, L., The influence of oxygen tension upon the
respiration of rat kidney slices, Arch. Biochem., 1948, 17, 61.
21. HILL, A. V., Diffusion of oxygen and lactic acid through tissues, Proc. Roy. Soc.
London, Series B, 1928, 104, 39.
22. LEE, K. S., Yu, D. H., and SAWYEH,P. N., unpublished data.
23. WOEHN~.R, C. A., Vasa Vasorum of Arteries. The Arterial Wall, Baltimore, The
Williams and Wilkins Co., 1959, 1.
24. OLSEN, N. S., and RUDOLPH, G. G., Transfer of sodium and bromide ions between blood, cerebrospinal fluid and brain tissues, Am. J. Physiol., 1955, 183,
427.
25. SI-IEATZ,G. C., and WILDE, W. S., Transcapillary exchange rate and volume of
distribution of sulfate and sodium as indicated by $3504 and Na 24 in the rat,
Am. J. Physiol., 1950, 162, 687.
26. W~SOLOWSKI,S. A., SAUVAGE,L. R., and PINC, R. D., Quantitative observations
during the freeze dry processing of arterial segments, J. Thoracic Surg., 1955,