102
Pressure-Induced Myogenic Activation of Cat
Cerebral Arteries Is Dependent on Intact
Endothelium
David R. Harder
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These studies were designed to determine the role of cerebral vascular endothelium in the "myogenic"
depolarization and contraction observed in isolated cat middle cerebral arteries exposed to high
transmural pressures. With intact endothelial cells we observed, on elevation of transmural pressure
in cannulated isolated arteries, significant membrane depolarization, action potential generation, and
reduction in internal diameter. After perfusion of the same vessels with collagenase and elastase for
short periods of time to disrupt the endothelial layer, all previous responses to elevation of transmural
pressure were no longer seen. Even though enzyme perfusion had no effect on membrane potential at
"control" levels of transmural pressure, it abolished the pressure-dependent depolarization, action
potential generation, and constriction. Furthermore, the contractile response to agonist stimulation
was maintained after endothelial disruption via enzymes, showing that this method of endothelial
disruption did not appreciably damage muscle cells. The data document a dependence of an intact
endothelium in mediating the activation of isolated cat cerebral arteries in response to a changing
transmural pressure. Thus, it is possible that the endothelial cell may serve as a transducer in the
autoregulatory response to pressure. (Circulation Research 1987;60:102-107)
C
erebral blood flow exhibits autoregulation over
a wide range of arterial blood pressures. We
have recently shown that when isolated cerebral arteries are cannulated and placed in an appropriate muscle myograph a pressure-dependent reduction
in internal diameter occurs, which is mediated by muscle cell membrane depolarization and action potential
generation.1>2 This action is not mediated by adventitial
nerves in that it occurs in the presence of neural
blockade.1
It was the purpose of this study to examine the role
of the endothelium in pressure-induced activation of
isolated cat cerebral arteries. With an intact, undisturbed endothelium, isolated middle cerebral arteries
exhibited membrane depolarization, action potential
generation, and reduction in diameter as observed previously. However, when the endothelium is disrupted
via perfusion of collagenase and elastase this same
pressure-dependent arterial muscle cell activation is no
longer observed. This lack of pressure-mediated tone
is not due to damage of the muscle cells by the enzyme
since the response to chemical stimulation is maintained. Thus, these data suggest that the endothelial
cell may serve as the transducer mediating changes in
transmural pressure to activation of cerebral arterial
muscle.
From the Departments of Neurology and Physiology, Medical
College of Wisconsin, Milwaukee, Wis., and Veterans Administration Medical Center, Milwaukee, Wis.
Supported by NIH grants 33833 and 31871, and the Veterans
Administration. Dr. Harder is an Established Investigator of the
American Heart Association and a Research Career Scientist of the
Veterans Administration.
Address for reprints: David R. Harder, PhD, VA Medical Center, Research/151, 5000 W. National Avenue, Milwaukee, WI
53295.
Received May 2 1 , 1986; accepted September 22, 1986.
Materials and Methods
Adult mongrel cats (2.0-4.0 kg of either sex) were
anesthetized with ketamine hydrochloride and sodium
pentpbarbital (30 mg/kg), decapitated, and brains were
removed. The left middle cerebral artery was dissected
free of arachnoid and placed in cold (4° C) physiological salt solution (PSS) consisting of (in mM) Na + 141,
Cl- 125, Ca 2+ 2.5, K + 4.7, Mg 2+ 0.76, H 2 PO 4 " 1.7,
HCO3~ 22.5, glucose 11, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 5.
HEPES was used as a partial buffer to control pH more
accurately.
One end of a 6-8 mm segment of middle cerebral
artery was threaded onto a 50-/u.m diameter plastic
cannula and tied in place with a 22-fx.m suture. The
opposite end was likewise cannulated. All side
branches were tied off with the above silk suture material. The cannulated artery was placed inside a muscle
myograph in which one cannula was fixed in plastic
jaws while the opposite end was connected to a micrometer so the vessel could be maintained at its in
vivo length. The inflow cannula was connected to a
pressure reservoir filled with PSS. Aligned between
the pressure reservoir and the arterial preparation was a
pressure transducer to monitor applied transmural
pressure. The system could either be flow-through by
leaving the outflow cannula patent or closed by clamping off the outflow cannula. The entire preparation was
suffused with PSS maintained at 37° C via a water
jacket. The PSS was aerated with 95% air and 5% CO 2 ,
yielding a Pco 2 of 38-40 torr and pH of 7.37-7.40.
Solution gases and pH were monitored via periodic
sampling measured with a Radiometer gas anaiyzer.
The PSS in the reservoir that perfused the vessel was
aerated and maintained identical to the suffusion solutions. Pressure measurements were made when the
Harder
Transducer for Myogenic Tone in Cerebral Arteries
system was closed so that it was not necessary to account for the flow resistance dk|he vessel. Internal
diameter was monitored witji|pr video system composed of a camera (RCA), TV monitor (Sony), and
VCR (Panasonic), and measured throughout the experiment with a Colorado Video, Inc., measuring system (Instruments for Physiology, Model 907, San
Diego, Calif.). Arterial dimensions were obtained 5
minutes after a step increase in luminal pressure of 20
mm Hg beginning at 20 mm Hg and ending at 160 mm
Hg. Magnification on the video screen was 150x.
Changes in diameter could be measured with an accuracy of ± 2 (im, which was the relative thickness of
the electronically produced lines generated by the
Colorado Video, Inc., measuring system.
Intracellular electrical activity was recorded with
103
glass microelectrodes using previously described techniques.1 Glass microelectrodes were filled with 3 M
KC1, had tip resistances of 50-80 Mft and tip potentials less than 3 mV. Criteria for successful impalements of arterial muscle cells have been described in
detail elsewhere.1 Measurements of electrical events
and internal diameter were made when the system was
closed so that specific values of transmural pressure
were accurately known.
The endothelium was disrupted by perfusing a PSS
containing 1 /ug/ml collagenase and 0.5 /tg/ml elastase
through the arterial segment for 3 minutes. After this
3-minute period, cold (20° C) PSS was perfused
through the vessel at 100 mm Hg to inactivate and
wash out enzymes. Efficacy of endothelial disruption
was assessed via electron microscopy. Figure 1 is an
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FIGURE 1. Electron
micrograph of a cat middle cerebral
artery section (5820 X magnification) before (panel A) and after
(panel B) perfusion of collagenase and elastase showing disruption of the endothelial surface
layer upon perfusion. Note the
tight endothelial-endothelial cell
junction (E).
104
Circulation Research
Vol 60, No 1, January 1987
ENDOTHELIUM INTACT
600
500
400
FIGURE 2. Panel A: Change in internal diameter in response to changing transmural pressure in isolated cannulated cat middle cerebral arteries. Due to the variability of
initial vessel dimensions at equilibrating (20 mm Hg) transmural pressure, the response in each of the 8 separate arteries studied is given. Note the tendency of vessels 2-7 to
decrease in internal diameter beyond 50-60 mm Hg transmural pressure. Vessels 1 and 8 maintained diameter from
60-120 mm Hg before becoming smaller beyond 120 mm
Hg. The endothelium in these cerebral arteries is intact.
Panel B: A graph depicting the same vessels as in Panel A
(vessel numbers are the same in both so all data are paired).
Note that after perfusion of cerebral arteries with collagenase and elastase to disrupt the endothelium, all 8 vessels
increased in diameter as a function of transmural pressure.
300
200
ENDOTHEUUM REMOVED
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600
B
500
INTERNAL
DIAMETER 400
(urn)
300
200
20
40
60
80
100
120
140
160
TRANSMURAL PRESSURE ( m m Hg)
electron micrograph (5,280 x ) from a vessel perfused
with enzymes showing disruption of the endothelial
layer (Figure IB), compared to a nonperfused vessel
showing intact endothelium with tight endothelial-endothelial cell junctions (Figure 1A). Details for electron microscopy are given in a separate communication.3 To test the viability of the muscle cells within the
artery after endothelial disruption with enzymes the
response to 30 mM KC1 and serotonin (3 X 10 ~7 M)
before and after perfusion were compared. In separate
experiments, we could never observe a dilatory response to acetylcholine in serotonin contracted arteries
when endothelium was disrupted in such a fashion with
enzyme perfusion (observed in 5 of 5 vessels).
All data in this manuscript are paired, i.e., the same
vessel is compared before and after endothelial disruption. Data were generated from cerebral arteries of 8
individual animals.
Results
Effect of Endothelial Disruption on Pressure-Mediated
Maintenance and Reduction of Internal Diameter
Internal diameter was measured as a function of
transmural pressure in 8 individual arteries. At trans-
mural pressures beyond 60 mm Hg the internal diameter was maintained or became smaller (Figure 2A). In
actual numbers, 2 preparations (1 and 8) maintained a
constant diameter in the face of an increasing pressure
load between 60 and 120 mm Hg and exhibited a
reduction in diameter only at pressures beyond 120
mm Hg. In the 6 other preparations depicted in Figure
2A, internal diameter gradually became less at each
pressure step beyond 50-60 mm Hg, which is consistent with autoregulatory ability, i.e., flow resistance
would increase at those higher transmural pressures.
Each point depicted in Figure 2 is the value of a single
diameter measurement taken 5 minutes after a 20 mm
Hg step increase in transmural pressure.
Conversely, disruption of the endothelium caused
all 8 arterial preparations to dilate passively as transmural pressure was elevated (Figure 2B). The numbers
in Figures 2A and 2B correspond to the same vessel
before and after endothelial disruption. The inability of
the vessels to respond to increased transmural pressure
with reduction in diameter did not appear to be a consequence of muscle cell damage during the enzyme perfusion since there was no significant difference in the
degree of vasoconstriction to high K+ or serotonin
before or after perfusion (in response to 30 mM KC1,
Harder
Transducer for Myogenic Tone in Cerebral Arteries
18 ± 4 % [SEM] of control diameter before vs.
24 ± 6% after enzyme perfusion; in response to
3 x 10" 7 serotonin, 28 ± 5% vs. 32 ± 8%). The integrity of the arterial muscle cell after endothelium disruption is further verified by the normal level of membrane potential, - 6 1 ± 2.4 mV (SEM) after enzyme
perfusion (see below). This increase in radial dimensions in the face of an increasing pressure load is most
likely completely passive since it occurs in the absence
of any changes in intracellular membrane potential
(Era) and is similar to that occurring when Ca 2+ influx
is blocked by verapamil, e.g., a mean diameter increase of 34% between 40-140 mm Hg in the absence
of endothelium vs. 38% in a separate set of experiments in the presence of verapamil (D.R. Harder and
J.A. Madden, unpublished observations).
105
-70
ENDOTHELIUM INTACT
•
•
•
•
-60
50
•
*J
* **
• •
•
• ••
POTENTIAL
— 40
-30
-20
1 ^
20
40
1
60
1B
H
80
1
^
100
1
•
1
120
1
•• • •
• •
•
1
140
1
160
ENDOTHELIUM REMOVED
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•
•
80
100
•
—60'
MEMBRANE
POTENTIAL
(mV)
-SO
—40
—30
—20
20
Comparison of Change in Diameter as a
Function of Change in Em
The diameter and Em data look at the pressure response in individual preparations with and without endothelium. Because of the variation in size of individual animals, there was a variation in size of any given
middle cerebral artery; the scale is large to accommodate such variation, and the differences in diameter
appear small. In an effort to compare more directly the
diameter response to increasing transmural pressure
with concomitant changes in Em, the diameter data
were normalized as percent change from control and
plotted against the change in Em. Resultant comparisons are depicted in Figure 4, which shows that in
these 8 cerebral arteries with normal intact endothelium there is a significant positive relation between an
increasing transmural pressure and reduction in internal diameter. The maximum reduction in internal diameter occurs at 160 mm Hg and is approximately
20%. There is also a significant correlation between
the observed reduction in diameter and level of membrane depolarization. The slope predicts a 1% reduction in internal radial dimension for a 1.9 mV reduction in Em. The meaning of such a relation will require
further study regarding cause and effect but does suggest that a membrane electrical mechanism may be
responsible for the myogenic response to pressure.
Any significant correlation between pressure and di-
•
•
-
MEMBRANE
—70
Inhibition of Pressure-Dependent Arterial Muscle Cell
Depolarization on Endothelial Disruption
Em was measured in 45 cell impalements from 8
arteries at various transmural pressures. As seen in
Figure 3, Em decreases as a function of transmural
pressure from a control value of - 6 3 ± 1 . 8 mV
(SEM) at 20 mm Hg to as low as - 22 mV at pressures
beyond 140 mm Hg. Disruption of the cerebral arterial
endothelium with collagenase and elastase prevented
this "pressure-dependent membrane depolarization"
(Figure 3B). Note, again, in Figure 3B that enzyme
perfusion in and of itself had no significant effect on
Em ( - 6 1 ± 2.4 mV at 20 mm Hg) at low transmural
pressures.
•
•
40
60
120
140
160
TRANSMURAL PRESSURE (mm Hg)
FIGURE 3. Panel A: Graph depicting membrane potential (Em)
measured with glass microelectrodes in 45 cells from 8 different
arteries measured as a function of transmural pressure with
intact endothelium. Each point is the value of a single impalement at that pressure. There is a linear relation between reduction ofEm.and elevation of transmural pressure with a correlation coefficient of 0.96. The Em at 20 mm Hg averaged
— 63 ±1.8 mV (SE). As can be seen, the Emfell as low as — 22
mVat pressures beyond 140 mm Hg. Panel B: Membrane potential (Em) as a function of transmural pressure after a 3-minute
perfusion with collagenase and elastase at 100 mm Hg perfusion pressure. Each point is an impalement at that pressure.
Note that enzyme perfusion did not significantly change Em at 20
mm Hg, demonstrating maintenance of cell integrity, and that
after enzymatic disruption of endothelium, there is no longer a
relation between Em and transmural pressure as observed in A.
ameter, and Em and diameter, is abolished upon disruption of the endothelium (Figures 2B and 3B).
Inhibition of Action Potential Generation in
Response to Elevated Transmural Pressure
on Endothelial Disruption
In 6 of 8 vessels studied, action potentials could be
recorded when transmural pressure exceeded 60-70
Circulation Research
106
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mm Hg. These action potentials were recorded only at
the base of side-branching arteries and were not observed for more than 1.5-2 mm on either side of a tied
off branch. Seen in Figure 5A (top) is a record of action
potentials recorded approximately 80 jinn from that
point where the cell in panel A (bottom) was impaled;
note how it appears to be propagated in that there is no
prominent prepotential such as that depicted in Figure
5A (bottom), which was recorded as close to the point
of bifurcation as possible. Even though this is an interesting finding, the point most relevant to this manuscript is that after disruption of the endothelium with
enzymes, no action potentials could be recorded from
the very same areas of the artery at the same transmural
pressure (Figure 5A vs. Figure 5B). However, in 8
preparations action potentials in areas visibly void of
branching arteries were not recorded. Note also, that
the membrane is hyperpolarized compared to the control after endothelial disruption. It should be noted that
in those cells that did not generate action potentials,
elevation in transmural pressure induced depolarization only, and Figure 3 represents data from all cells.
In spiking cells, the level of Em was taken as the most
hyperpolarized state.
100
95-
INTERNAL
DIAMETER
9085-
75
20
-
100 1
40
60
80
100
120
TRANSMURAL PRESSURE (mm Hg)
100
B
^_
40
^ ^ 5
(mmHg)
SO
N. 100
% CHANGE
IN
90
INTERNAL
DIAMETER
«^^
R = 0.98
^^S^
120
140
^ \ _
80
10
20
30
CHANGE IN MEMBRANE POTENTIAL (mV)
40
FIGURE 4. Representation of normalized data from Figure
2A. Data is normalized as percent of diameter from control
(i.e., 100% is control) at each transmural pressure. Regression
analysis depicts a positive relation between pressure and reduction of diameter (negative slope), with a correlation coefficient
ofO. 91. In Panel B, the normalized diameter data is plotted as a
function of change in Em at each transmural pressure, the slope
of which indicates a 1.9 mV change in Em for each 1% change
in internal diameter and a highly significant correlation coefficient of 0.98. The solid unsymboled lines in A and B represent
the calculated slope obtained from regression analysis.
TRANSMURAL
Vol 60, No 1, January 1987
PRESSURE =120 mmHg
A,Intact Endothelium
1
A , I n t a c t Endothelium
r°
—V
i
i
i
i
i
n
EmV
m,
-60
Tsec
Ssec
'"Isee
4sec
4lei
sec
FIGURE 5. Actual chart record depicting spontaneous action
potentials fPanel A, top and bottom) recorded at branch points
in isolated arteries exposed to 120 mm Hg transmural pressure.
Panel B (top and bottom) represents cell impalements in the
exact area after perfusion ofcollagenase and elastase to disrupt
the endothelium. Apart from the inhibition of action potential
generation after endothelial disruption, note also that the E „ is
more polarized. This figure depicts action potentials recorded
at a point of bifurcation (A, bottom) between the main artery
and a branch demonstrating prominent pacemaker activity. The
action potentials in the top of panel A were recorded 80 yjn
away from that point impaled in the bottom record of the same
panel. The absence of prominent pacemaker prepotentials suggests that the action potentials may be propagated; no action
potentials were recorded greater than 2 mm away from the point
impaled in Panel A (bottom). All impalements were from the
same vessel.
Discussion
Much has been written about the influence of substances released from the endothelial cell layer on the
muscle cells within the same arteries. There is little
direct evidence identifying the nature and/or composition of those endothelial derived substances; however,
the action of these agents appears most often to be
dilatory.4 In some arterial beds and under certain conditions, constrictor substances that are released from
vascular endothelium can also be identified.5"7 A recent abstract also demonstrates a constrictor substance
released from dog cerebral arteries upon stretch.8
The data presented here are in precise agreement
with those of Katusic et al. 8 An intact endothelium
does indeed appear to be necessary in order to elicit the
membrane depolarization, action potential generation,
and pressure-dependent reduction of internal diameter
in isolated cat cerebral arteries. The absence of pressure-mediated excitatory events does not appear to be
due to damage of cerebral arterial muscle upon perfusion ofcollagenase and elastase in that 1) the response
to chemical agonists is not altered by the treatment;
and 2) a stable Em not different from control after
Harder
Transducer for Myogenic Tone in Cerebral Arteries
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enzyme perfusion is maintained, showing that the
muscle cell membrane was not damaged. Furthermore, the dilatory action of transmural nerve stimulation is not affected by enzyme perfusion in cat middle
cerebral arteries.3
It appears that vascular endothelial cells, at least in
cat and dog cerebral arteries, may serve a "transducer
function" in that a mechanical force liberates a chemical mediator that can activate the adjacent arterial
muscle cells. The increase in radial dimensions upon
elevation of transmural pressure in the absence of endothelium is most likely a passive phenomenon, since
there is no change in Em and increases in a linear
fashion with pressure. A similar passive phenomenon
is observed in cat cerebral arteries upon inhibition of
Ca2+ influx with verapamil.9 The data in the present
study suggest that an increase in transmural pressures
creates a stress across the endothelial cell that activates
the release of a chemical mediator. It is not possible at
this point to determine the specificity of the type of
mechanical stimulus, i.e., the sheer stress that would
occur as transmural pressure increased, actual stretching of the cell surface, or a point source of pressure
analogous to activation of a pacinian corpuscle. What
is clear from this study is that the chemical mediator(s)
act via electromechanical coupling initiating a change
in ion conductance, which then activates the muscle
cell. Indeed, this entire process can be inhibited by
blocking Ca 2+ or increasing K + conductance 19 (unpublished observations).
It is intriguing that action potentials are not universally recorded along the entire surface of a pressurized
artery, but only at the bifurcation points of branching
arteries. Obviously, this has important implications
107
which require further independent study before any
conclusions can be drawn regarding the function of
these spontaneously active cells. It is of merit, however, to point out that an intact endothelium is required to
initiate regenerative electrical activity in these cells.
References
1. Harder DR: Pressure-dependent membrane depolarization in cat
middle cerebral artery. Circ Res 1984;55:197-202
2. Harder DR, Lombard JH: Voltage-dependent mechanisms of
receptor stimulation in cerebral arterial muscle, in Bevan JA,
Godfraind T, Maxwell RA, Stoclet JC, Worcel M (eds): Vascular Neuroeffector Mechanisms. Amsterdam, The Netherlands,
Elsevier, 1985; pp 181-186
3. Harder DR, Madden JA: Electrical stimulation of the endothelial
surface of pressurized cat middle cerebral arteries results in
TTXTsensitive vasoconstriction. Circ Res 1987 (in press)
4. Furchgott RF: Role of endothelium in response to vascular
smooth muscle. Circ Res 1983;53:557-573
5. Hickey KA, Rubanyi G, Paul RJ, Highsmith RF: Characterization of a coronary vasoconstriction produced by cultured endo%
thelial cells. Am J Physiol 1985;248:C550-C556
6. Gabor M, Vanhoutte PM: Hypoxia releases vasoconstrictor substances from the coronary endothelium (abstract). Circulation
1984;70:122
7. O'Brien RF, McMurtry IF: Endothelial cell supernates contract
bovine pulmonary artery rings (abstract). Am Rev Respir Dis
1984;192:337
8. Katusic ZS, Shepherd JT, Vanhoutte PM: Endothelial-dependent contraction to stretch in canine basilar arteries (abstract).
Fed Proc 1986;45:289
9. Lombard JH, Smeda J, Madden JA, Harder DR: Effect of reduced oxygen availability upon myogenic depolarization and
contraction of cat middle cerebral artery. Circ Res 1986;58:565569
KEY WORDS
physiology •
cerebral arteries • endothelium
myogenic activation
electro-
Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact
endothelium.
D R Harder
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Circ Res. 1987;60:102-107
doi: 10.1161/01.RES.60.1.102
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1987 American Heart Association, Inc. All rights reserved.
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