Adrenergic Neurotransmission in Tail Arteries from
Two-Kidney, One Clip, Renal Hypertensive Rats
R. CLINTON W E B B , P H . D . , JOYCE C. JOHNSON, B.S.,
DAVID F. BOHR,
AND
M.D.
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SUMMARY The goal of this study was to determine if increased vascular smooth muscle sensitivity
to norepinephrine in two-kidney, one clip (2K1C) hypertensive rats is the result of a decrease in
adrenergic nerve function. Vascular sensitivity to norepinephrine was measured in isolated tail artery
strips from 2K1C hypertensive and normotensive rats and in various arterial strip preparations from
normotensive rats that exhibit varying degrees of adrenergic innervation. In each case, the characteristic of the vascular smooth muscle response in the vessel with the least amount of adrenergic innervation simulated the response of the vascular smooth muscle from the 2K1C hypertensive rats. Release
or displacement of endogenous norepinephrine by electrical stimulation, tyramine, potassium-free
solution, and potassium excess, and measurement of tissue content of norepinephrine suggest that the
blood vessels of 2K1C hypertensive animals are depleted of catecholamine stores. Based on these
observations it is concluded that the increased sensitivity of vascular smooth muscle to norepinephrine
in 2K1C hypertensive rats is the result of a diminished adrenergic innervation. This increased
sensitivity of the vasculature may be a response of the smooth muscle cells to a decrease in innervation
or the consequence of vascular wall hypertrophy leading to an increased number of smooth muscle
cells that are remote from their adrenergic supply.(Hypertension 5:298-306, 1983
KEY WORDS * aorta • mesenteric artery * acute and chronic adrenergic denervation
norepinephrine • cocaine * vascular sensitivity
T
rats and dogs. Interpretation of the significance of this
catecholamine depletion in terms of vascular function
has been speculative. Most authors"' l 2 postulate that
increased norepinephrine turnover and depleted nerveending catecholamine stores provide evidence of an
increased sympathetic influence on the vasculature.
This increased sympathetic influence could be due to
either an augmented sympathetic nerve activity or increased transmitter release at a normal level of sympathetic activity. Facilitation of norepinephrine release
from adrenergic nerve endings by angiotensin II, liberated following renal artery clipping, also has been suggested as a possible means whereby normal levels of
sympathetic activity could enhance vasoconstriction.8
The possibility that catecholamine depletion might imply reduced adrenergic nerve function has received
little consideration.'
The goal of this study was to determine if increased
vascular smooth muscle sensitivity to norepinephrine
in two-kidney, one clip (2K1C) hypertensive rats is the
result of a decrease in adrenergic nerve function. Since
somewhat similar changes in vascular sensitivity to
catecholamines occur following chronic adrenergic
denervation,13 vascular responsiveness of normotensive arteries following destruction of adrenergic nerves
was determined. Additionally, vascular responsive-
HE junction between the adrenergic nerve ending and the smooth muscle cells of the blood
vessel wall makes sympathetic control of vascular function possible. Experimental evidence developed over the past two decades may be interpreted as
indicating that functional changes in this nerve-muscle
relationship contribute to the increase in total peripheral resistance of experimental renal hypertension.1"3
Specifically, both catecholamine content4"6 and catecholamine histofluorescence7-8 have been found to be
significantly decreased in vascular beds of animals
with both renal clip hypertension and renal hypertension induced by figure-eight parenchymal ligature
technique. Ultrastructural studies9-10 indicate a decrease in the number of dense core vesicles in adrenergic nerve endings of arteries from renal hypertensive
From the Department of Physiology, University of Michigan,
Ann Arbor, Michigan.
Supported by a grant from the Michigan Heart Association and
Grants (HL-18575 and HL-27020) from the National Institutes of
Health. Dr. Webb is the recipient of Research Career Development
Award HL-OO813 from the National Institutes of Health.
Address for reprints: Dr. R. Clinton Webb, Department of Physiology, University of Michigan, 7720 Medical Science Building II,
Ann Arbor, Michigan 48109.
Received May 26, 1982; revision accepted November 11, 1982.
298
ADRENERG1C INNERVAT10N IN RENAL HYPERTENSION/WeM? et al.
ness of a densely innervated blood vessel (tail artery;14- 15) was compared to that of the aorta which is
virtually devoid of an adrenergic supply. 16 ' l7 Vascular
sensitivity to norepinephrine of the mesenteric artery
was determined since this vessel is intermediate in its
density of innervation.17
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Methods
Animal Preparation
All studies were performed on male, Sprague-Dawley rats (n = 40; 400-475 g body weight at the time of
experimentation). Twelve of the rats were anesthetized
with ether, and the left kidney was exposed through a
flank incision. A silver clip (0.22 mm slit) was placed
on the left renal artery. Normotensive rats did not
undergo sham treatment. All rats were maintained on
standard laboratory chow (Purina) and tap water ad
libitum. Systolic blood pressures were determined in
the conscious state by means of indirect tail cuff measurements. Experiments were performed at 4 months
after surgery.
299
in a bicarbonate-free PSS containing 300 /ig/ml 6hydroxydopamine for 10 minutes. The pH of this unbuffered PSS was adjusted to 4.0 by the addition of 20
piM glutathione. The O2-CO2 mixture to the muscle
bath was turned off during the denervation procedure.
Following denervation, the strips were allowed to recover in normal PSS for 3 hours.
Chronic denervation of the tail artery was performed
in six normotensive rats.18 Each rat was anesthetized
with ether, and a 10 mm portion of the artery was
exposed at the origin of the tail. The surface of the
vessel was mechanically scraped with serrated forceps
and the artery was painted for 5 mm of its length with
10% phenol in ethanol. Two to 3 weeks after surgery,
the rats were terminated by a blow to the head, and the
tail artery distal (1-2 cm) to the treatment area was
excised and cut into helical strips as described above.
Norepinephrine Measurements
The tissue content of norepinephrine in tail arteries
and the plasma levels of the catecholamine were determined radioenzymatically using a commercially available kit (Cat-a-Kit, Upjohn Diagnostics) as described
by Vanhoutte et al.19
Arterial Strip Preparation
All rats were terminated by a blow to the head, and
aortas, mesenteric arteries, and/or tail arteries excised.
The arteries were stored in physiological salt solution
(PSS) and cut helically into strips (1.0 X 10 mm)
under a dissecting microscope. The helical strips were
mounted vertically on either a glass or plastic holder in
a tissue bath containing PSS. The upper ends of the
strips were connected to force transducers (Grass
FT.03), and the resting force placed on each strip was
adjusted so that the strip produced maximum active
force in response to a standard dose of norepinephrine
(10~7 g/ml). The bathing medium was maintained at
37°C and aerated with a mixture of 95% O2 and 5%
CO2. The pH of the solution was 7.4 and the composition (mmole/liter) was as follows: NaCl (130), KC1
(4.7), KH2PO4 (1.18), MgSGy7H2O (1.17), CaC12«2H2O (1.6), NaHCOj (14.9), dextrose (5.5), and
CaNa2EDTA (0.03). Potassium-free solution was of
the same composition except that KC1 was omitted and
1.2 mM KH2PO4 was substituted with 1.2 mM
NaH2PO4. Higher concentrations of potassium (10 to
130 mM) in the bathing medium were achieved by
equimolar substitution of NaCl with K G . Before the
start of experiments, the strips were allowed to equilibrate for 90-120 minutes in PSS.
Arterial strips were electrically stimulated by the use
of two platinum wire electrodes placed parallel to the
preparations. Electrical impulses consisted of square
waves (9 V, 2.0 msec) provided by a direct current
power supply and switching transistor triggered by a
stimulator (Grass, Model S4E).
Results
At the time of experimentation (4 months after surgery), the systolic blood pressures of rats used in this
study were: 2K1C hypertensive = 198 ± 3 mm Hg (n
= 12) and normotensive = 121 ± 2 m m H g ( n = 22;
p < 0.05). Chronic denervation of the tail artery did
not alter the systolic blood pressure of normotensive
rats (118 ± 3 mm Hg; n =; 6).
Acute and Chronic Denervation Procedures
In some experiments, helical strips of tail artery
from hypertensive and normotensive rats were acutely
denervated with 6-hydroxydopamine according to the
method of Aprigliano and Hermsmeyer.14 Following a
90-minute equilibration period, the strips were placed
Passive Force and Contractile Responses to Norepinephrine
Before the start of the experiments, the arterial strips
were stretched to successively greater levels of passive
force. At each 100 mg increment of passive force applied to the strips, contractile responses to 10" 7 g/ml
norepinephrine were determined. The optimum pas-
Statistical Methods
The results of these experiments were analyzed by
several statistical procedures. The responses of each
arterial strip were normalized to its maximal response
to exogenous norepinephrine to allow interpretation of
the results in terms of vascular reactivity and sensitivity. Dose-response and frequency-response curves
were calculated as geometrical means. Paired and unpaired t tests and curve fitting analyses (logit-log transformation) were performed. A p value less than 0.05
was considered to be statistically significant.
Drugs
Drugs used were: norepinephrine bitartrate (Breon
Laboratories, Inc.), 6-hydroxydopamine hydrobromide (Sigma Chemical Company, St. Louis, Missouri), glutathione (Sigma Chemical Company),
tyramine (Sigma Chemical Company), and cocaine
(supplied by University of Michigan Hospital Pharmacy, Ann Arbor, Michigan).
300
HYPERTENSION
sive force for generation of active contractile responses
was defined as the passive force that gave no further
increase in active force generation for two successive
100 mg increments. In tail arteries from 2K1C hypertensive and normotensive rats, contractile responses to
norepinephrine increased to a maximum as the passive
force applied to the strips increased to approximately
500 mg. The optimum passive force for maximum
response to norepinephrine was similar for strips from
2K1C hypertensive rats (634 ± 42 mg; n = 12) and
those from normotensive rats (608 ± 37 mg; n = 22).
The optimum passive force for aortic strips was 1425
± 138 mg (n = 6) and that for mesenteric arteries was
542 ± 24 mg (n = 6). The chronic denervation procedure did not alter this passive force-active force relationship (optimum passive force in chronically denervated tail arteries = 637 ± 35 mg, n = 6).
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The dose-response curve for norepinephrine was
shifted to the left in tail artery segments from 2K1C
hypertensive rats with respect to that obtained in tail
arteries from normotensive rats (left panel, fig. 1). The
dose of norepinephrine which caused a half-maximal
response (ED^) was significantly less in tail arteries
from hypertensive rats (2.2 X 10"9g/ml) as compared
to that in tail arteries from normotensive rats (10.8 x
10~9g/ml; p < 0.05). The maximal contractile force
developed was significantly less for arterial strips from
hypertensive rats (1792 ± 112 mg) as compared to
that developed by tail arteries from normotensive rats
(2183 ± 117 mg;p < 0.05).
Comparison of sensitivity to norepinephrine in three
different arteries from normotensive rats (middle panel, fig. 1) indicated that aortic strips were most sensitive to the catecholamine (ED*, = 0.4 X 10~9 g/ml);
whereas tail arteries were the least sensitive (ED^ =
10.8 X 10"9 g/ml); mesenteric arteries were intermediate in sensitivity to norepinephrine (ED^ = 4.5 x
10"9 g/ml). Maximal contractile force developed in
response to norepinephrine was: aorta = 967 ± 62
mg; mesenteric artery = 672 ± 42 mg; tail artery =
2183 ± 117 mg).
There was a significant shift to the left in the doseresponse relationship for norepinephrine following
chronic denervation of the tail artery in normotensive
rats (right panel, fig. 1). The ED^ value for norepinephrine was significantly lower in chronically denervated arterial strips (2.7 x 10~9g/ml) than in innervat-
Dose Response to Norepinephrine
Cumulative addition of norepinephrine (10~12 to
10"5 g/ml) to the muscle bath produced contractile
responses in all arterial strips (fig. 1). Experimental
observations for tail arteries from normotensive rats,
which did not undergo the surgical denervation procedure, were averaged and analyzed together (i.e., the
tail arteries labeled as "normotensive" in the left panel
of figure 1 are the same as those labeled as "tail artery" and "innervated" in the middle and right panels
of figure 1, respectively; this averaging process was
used in subsequent analyses in figures 2 and 5).
2K-IC Hyp»rUn«ion - tail arttfy
V O L 5,
IQTIO
Tail Arttry - chronic derivation
, 0 -6
[ Norepinephrine ]
(g/ml)
FIGURE 1. Dose-response to norepinephrine. Helical strips of aortas and mesenteric and tail arteries from 2KIC renal hypertensive
or normotensive rats were made to contract in response to the cumulative addition of norepinephrine to the muscle bath. Left panel:
Tail arteries from hypertensive rats were more sensitive to the catecholamine than those from normotensive rats. Middle panel: Tail
arteries were less sensitive to norepinephrine than mesenteric arteries or aortas. Right panel: Denervation of the tail artery resulted
in an increased sensitivity to norepinephrine. Asterisks indicate statistical differences between arteries from hypertensive rats and
those from normotensive rats (left panel), differences between tail arteries and aortas or mesenteric arteries (middle panel), and
differences between innervated and chronically denervated tail arteries (right panel, p < 0.05). Values are the means ± standard
error of the mean (SEM). The values in parentheses are the number of rats.
ADRENERGIC INNERVATION IN RENAL HYPERTENSION/WfeM et al.
ed control arterial strips (10.8 x 10"9 g/ml; p <
0.05). Maximal contractile responses to norepinephrine were less in denervated tail arteries (1337 ± 202
mg) than in innervated tail arteries (2183 ± 117 mg;p
< 0.05).
teric arteries were intermediate in responsiveness to
field stimulation (middle panel, fig. 2). Maximal contractile responses were: tail arteries = 1681 ± 128 mg
(n = 8); mesenertic arteries = 260 ± 35 mg (n = 6);
aortas = 16 ± 16 mg (n = 6).
After chronic denervation of the tail artery, contractile responsiveness to electrical stimulation of adrenergic nerve endings was depressed compared to control
innervated tail artery segments (right panel, fig. 2).
Maximal contractile responses were: innervated =
1681 ± 128 mg (n = 8); and denervated = 547 ±
125 mg (n = 4). These observations suggest that the
surgical technique produced a partial denervation of
the tail artery.
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Frequency-Response Relationship
Cumulative frequency-response curves in all arterial
preparations were performed by starting electrical
stimulation at 0.25 Hz; the frequency was increased
stepwise to 0.5, 1, 2, 4, 8, 16, and 32 Hz when the
contractile response to the previous stimulation frequency had reached a maximum (fig. 2). Contractile
responses at each frequency of stimulation reached a
plateau in 10 to 20 seconds after the stimulation frequency was changed. Contractile responses to electrical stimulation were abolished by 10~6 M phentolamine and absent in arterial strips acutely denervated
with 6-hydroxydopamine.
Contractile responses to electrical stimulation in tail
artery strips from 2K1C hypertensive rats were less at
all frequencies of stimulation than those in arterial
strips from normotensive rats (left panel, fig. 2). Maximal contractile responses were: 2K.1C hypertensive
rats = 860 ± 169 mg (n = 8); normotensive rats =
1681 ± 128 mg (n = 8).
Comparison of contractile activity to electrical field
stimulation indicated that tail arteries were the most
responsive to activation of adrenergic nerve endings,
whereas aortic strips were the less responsive; mesen-
I
2K-IC
Hypertension - toil artery
Vessel
Dose Response to Tyramine
Cumulative addition of tyramine (10" 7 to 10"2 M) to
the muscle bath produced contraction in tail artery
strips from 2K1C hypertensive and normotensive rats
(fig. 3). Innervated arterial strips from hypertensive
rats responded similarly to tyramine as those from normotensive rats. Statistical differences (unpaired / test)
were evident at only two doses of tyramine (3 X 10"6
M and 10~3 M). Acute denervation with 6-hydroxydopamine reduced the contractile effect of tyramine;
contractile responsiveness of tail artery strips from hypertensive rats were less affected by destruction of
adrenergic nerve endings than those from normotensive rats as indicated by the smaller shift to the right
and depression of the maximal response to tyramine
Com pan ton
Tail
*
100
00
100
§
80
80
80
«
60
60
|
40
»
20
I°
tan a
(N-8)
40
*
(N-8)
, ' ' mMtntftc artery
20
0
0.25
Artery - chronic denervation
60
40
1
301
0
••'
0.25
I
4
Frequency
16
0.25
(Hz)
FIGURE 2. Frequency-response relationship. Helical strips of aortas and mesenteric and tail arteries from 2KIC renal hypertensive
or normotensive rats were made to contract in response to electrical field stimulation of adrenergic nerve endings. Left panel: Tail
arteries from hypertensive rats contracted less in response to field stimulation than those from normotensive rats. Middle panel:
Aortas contracted less in response to field stimulation than tail and mesenteric arteries. Right panel: Tail arteries which were
chronically denervated contracted less in response to field stimulation than control innervated tail arteries. Asterisks indicate
statistical differences between arteries from hypertensive rats and those from normotensive rats (left panel), differences between tail
arteries and aortas or mesenteric arteries (middle panel), and differences between innervated and chronical7v denervated tail arteries
(right panel, p < 0.05). Values are means ± SEM. The values in parentheses are the number of rats.
HYPERTENSION
302
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1
5
3, MAY-JUNE
1983
Contractile responses to 10 4 M tyramine were depressed by treatment with 10~6 M cocaine in tail arteries from both normotensive and 2K1C hypertensive
rats (right panel, figure 4). The magnitude of the depression of the percent contractile response was greater
(untreated values minus values obtained in the presence of cocaine) in tail arteries from normotensive rats
(-26%) than in those from hypertensive rats
(-13%).
innervated
60
VOL 5, No
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I
acute
denervotion
M, EK-IC
20
(N-6)
r r
I
IO' 7
normo
*V
icr5
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[Tyramine]
—• normoloniivt
iA~]
io(M)
FIGURE 3. Dose-response to tyramine. Helical strips of tail
arteries from hypertensive and normotensive rats were made to
contract in response to the cumulative addition of tyramine to
the muscle bath. Dose-response curves were performed before
and after acute denervation with 6-hydroxydopamine. Asterisks
indicate statistical differences between the innervated and
acutely denervated state fp < 0.05). Daggers indicate statistical differences between hypertensive and normotensive rats (p
< 0.05). Values are the means ± SEMfor six normotensive rats
and six hypertensive rats.
following treatment with 6-hydroxydopamine. Maximal contractile responses to tyramine were: 2K1C hypertensive rats, innervated = 1022 ± 132mg(n = 6);
normotensive rats, innervated = 1418 ± 231 mg (n
= 6); 2K1C hypertensive rats, denervated = 549 ±
147 mg (n = 6); normotensive rats, denervated = 267
± 85 mg (n = 6).
Cocaine, Acute Adrenergic Denervation and Contractile
Responses to Norepinephrine, Electrical Field
Stimulation, and Tyramine
Cocaine (10~6 M), a neuronal uptake inhibitor, and
acute denervation with 6-hydroxydopamine potentiated contractile responses to exogenous norepinephrine
in tail artery strips from normotensive rats, whereas
contractile responsiveness to the catecholamine was
not significantly altered by these interventions in tail
arteries from 2K1C hypertensive rats (left panel,
fig. 4).
Treatment of tail artery strips from normotensive
rats with 10"6 M cocaine potentiated contractile responses to 2 Hz electrical stimulation of adrenergic
nerve endings; contractile responses to 8 Hz stimulation were not altered by the neuronal uptake inhibitor
(middle panel, fig. 4). Cocaine had no effect on contractile responses to field stimulation in tail arteries
isolated from 2K1C hypertensive rats.
Contraction in Response to Potassium-Free Solution
Exposure of arterial strips to potassium-free solution
resulted in contraction (fig. 5). These contractions
were slow in development, reaching a maximum at 15
to 20 minutes after exposure to the potassium-free solution. The contractions were absent in arterial strips
acutely denervated with 6-hydroxydopamine and abolished by 10~6 M phentolamine. Contractile responses
of tail artery strips from 2K1C hypertensive rats were
significantly less than those from normotensive rats at
15, 20, 25, and 30 minutes into the potassium-free
cycle (left panel, fig. 5). Tail artery segments from
normotensive rats contracted the most in response to
potassium-free solution, aortic strips contracted the
least, and mesenteric arteries were intermediate in responsiveness to the absence of potassium (middle panel, fig. 5). Chronic surgical denervation of tail arteries
reduced the contractile effect of potassium-free solution (right panel, fig. 5). Maximal contractile responses were: 2K1C hypertensive rats = 633 ± 163
mg (n = 6); normotensive rats = 1268 ± 128 mg (n
= 10); aortas = 126 ± 49 mg (n = 6); mesenteric
arteries = 138 ± 38 mg (n = 6); denervated tail
arteries = 480 ± 101 mg (n = 6).
Dose Response to Potassium
Tail artery strips from 2K1C hypertensive and normotensive rats were made to contract in response to the
cumulative addition of potassium to the muscle bath in
the presence and absence of 10^M phentolamine (fig.
6). In the absence of phentolamine, arterial strips from
2K1C hypertensive rats and normotensive rats responded similarly. Phentolamine (10~6 M) reduced the
contractile effect of elevated potassium; contractile responses to potassium in arterial strips from hypertensive rats were less affected by alpha adrenergic blockade than were those from normotensive rats. Maximal
contractile responses to potassium were: for 2K1C hypertensive rats, untreated = 1023 ± 122mg(n = 6);
for normotensive rats, untreated = 1666 ± 154mg(n
= 6); for 2K1C hypertensive rats, phentolamine =
702 ± 74 mg (n = 6); and for normotensive rats,
phentolamine = 848 ± 75 mg (n = 6).
Plasma Levels and Tissue Content of Norepinephrine
Plasma levels of norepinephrine were similar in hypertensive and normotensive rats, whereas the tissue
content of the catecholamine was significantly less in
tail artery strips isolated from hypertensive rats than in
those from normotensive rats (fig. 7).
303
ADRENERGIC INNERVATION IN RENAL HYPERTENSION/Webb et al.
Norepinephrine
I
I
I
Field
100
Stimulation
Tyramine
100
100
80
80
60
60
60
40
40
40
20
20
20
80
0
D
Untnottd
0
K)"6 M Cocain*
•
Acuta tftntrvation
KT» g/ml
0
3 i K>» o/ml
2 Hz
8 Hi
0
2HI
2K-IC
(N>6)
2K-IC
(N>6)
K>'«M
Kr* M
2K-IC
(N-8)
(N-6)
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FIGURE 4. Cocaine, acute adrenergic denervation and vascular responses to norepinephnne, field stimulation, and tyramine.
Helical strips of tail arteries from hypertensive and normotensive rats were made to contract in response to norepinephrine, electrical
field stimulation, and tyramine. Left panel: Cocaine (IO~6) and acute denervation potentiated vascular responses to norepinephrine in
arterial strips from normotensive rats but not those in arterial strips from hypertensive rats. Middle panel: Contractile responses of
normotensive arterial strips to 2 Hz stimulation were potentiated in the presence of 10~6 M cocaine; contractile responses to 2 Hz in
hypertensive arterial strips and to 8 Hz in both normotensive and hypertensive strips were not altered by IO~6 M cocaine. Right
panel: Cocaine (IO~6 M) inhibited contractile responses to 10~4 M tyramine to a greater degree in arterial strips from normotensive
rats than in those from hypertensive rats. Asterisks indicate statistical differences between untreated and treated condition (p<0.05).
Values are means ± SEM for six normotensive rats and six hypertensive rats.
2K-IC
!
Companion
Toil
100
100
100
80
80
80
60
60
-I—t
40
i
I
I
Vetsel
Hypertension - toil ortery
20
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(N-IO)
Artery - chronic
denervation
60
40
40
20
20
2K-IC (N'6)
IS
25
15
Minutes
15
25
in Potassium - free
25
Solution
FIGURE 5. Contraction in response to potassium-free solution. Helical strips of aortas and mesenteric and tail arteries from 2K1C
renal hypertensive or normotensive rats contracted when placed in potassium-free solution. Left panel: Tail arteries from hypertensive rats contracted less in response to potassium-free solution than those from normotensive rats. Middle panel: Aortic strips
contracted less than mesenteric and tail arteries. Right panel. Chronically denervated tail arteries contracted less than control
innervated tail arteries. Asterisks indicate statistical differences between arteries from hypertensive rats and those from normotensive
rats (left panel), differences between tail arteries and aortas or mesenteric arteries (middle panel) and differences between innervated
and chronically denervated tail arteries (right panel, p<0.05). Values are means ± SEM. The wlues in parentheses are the number of
rats.
304
HYPERTENSION
VOL 5, No
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1983
Plasma
80
Untreated
It
1
^
60
300
5 200
Norepine/.
40
20
o
o
11.1
0
[Potassium
Chloride] (mM)
•
Toil Artery
I
10
i
T
•^ 8
1 I
1
Normotensive
2K-IC
*
I
.
I
i
(N»6)
(N=6)
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FIGURE 6. Dose-response to potassium. Helical strips of tail
arteries from hypertensive and normotensive rats were made to
contract in response to the cumulative addition of KCl to the
muscle bath. Dose-response curves were performed before and
after treatment with 10~6 M phentolamine. Asterisks indicate
statistical differences between untreated strips and those treated with 10~6 M phentolamine (p < 0.05). Daggers indicate
statistical differences between hypertensive and normotensive
rats (p < 0.05). Values are means ± sEMfor six normotensive
rats and six hvpertensive rats.
FIGURE 7. Plasma and tissue content of norepinephrine. The
norepinephrine content of plasma samples and tail arteries
from hypertensive and normotensive rats were determined by
radioenzymatic assay. There was no statistical difference in the
plasma levels of the catecholamine between hypertensive and
normotensive rats whereas the tissue content of norepinephrine
was lower in tail arteries from hypertensive rats compared to
that in tail arteries from normotensive rats. The asterisk indicates a statistical difference between hypertensive and normotensive rats (p < 0.05). Values are means ± SEM for six
normotensive rats and six hypertensive rats.
Discussion
Extensive evidence from broadly different types of
experiments support the hypothesis that the sympathetic nervous system is involved in the development of
renal hypertension.1"12 The current study uses two different systems to evaluate the effect that adrenergic
innervation has on vascular smooth muscle responsiveness. The effect of the naturally occurring variability in adrenergic nerve density of three different blood
vessels (aorta, mesenteric artery, tail artery) was studied. In the second system, the effect of chronic surgical
denervation was evaluated. In both of these systems,
the characteristic of the vascular smooth muscle response in the vessel with the least amount of adrenergic innervation simulated the response of the vascular
smooth muscle from the 2K1C hypertensive rat.
to that observed in tail arteries from normotensive rats
which had been partially denervated by surgical procedure (right panel, fig. 1). Additionally, it was observed
that the variability of adrenergic nerve density in aortae, mesenteric arteries and tail arteries correlated inversely with sensitivity to norepinephrine. The aorta
which is virtually devoid of adrenergic innervation,
based on its catecholamine content (~0.2 fig/g wet
weight)17 was the most sensitive to norepinephrine
(lowest ED^), whereas the tail artery, which has the
highest catecholamine content (8.5 fxg/g wet weight;
fig. 7), was the least sensitive to the catecholamine.
Mesenteric arteries were intermediate in sensitivity to
the catecholamine, and these arteries contain intermediate levels of norepinephrine (6.0 fig/g wet weight)."
This similarity in performance of vascular smooth
muscle in 2K1C hypertension and that of poorly innervated muscle from a normotensive animal was evident
also in the maximal force developed by tail artery
strips from the respective groups. The maximal force
developed in response to norepinephrine was less in
tail artery strips from 2K1C hypertensive rats than in
those from normotensive rats; and the maximal force
developed by chronically denervated tail arteries was
less than control innervated tail arteries. Other investigators have observed a similar decreased maximum
force generating ability in arterial strips from hypertensive animals21"25 and in vascular smooth muscle which
Vascular Sensitivity to Norepinephrine
Vascular sensitivity to norepinephrine was increased in isolated tail arteries from 2K1C hypertensive rats as evidenced by the leftward shift in the doseresponse relationship relative to that in tail arteries
from normotensive rats (fig. 1). Other investigators
have observed a similar increased sensitivity to the
catecholamine in perfused vascular beds and isolated
blood vessel preparations from renal hypertensive animals (see ref. 20 for review). This increased sensitivity
to norepinephrine in hypertension is strikingly similar
305
ADRENERGIC INNERVATION IN RENAL HYPERTENSlON/WeW? et al.
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has been chronically denervated. l3 The reasons for thir
difference in force generating ability between vascular
smooth muscle from hypertensive animals and that
from normotensive animals is not clear, but it does not
appear to be related to the resting force placed on the
arterial strips.13 • 2 3 " Berner et al.26 induced vascular
hypertrophy by partial ligation of the rabbit portalanterioir mesenteric vein. At 14 days after ligation
there was a massive increase in the intermediate filaments surrounding the dense bodies in which actin
filaments insert. The proliferation of intermediate filaments was concomitant with a decrease in number and
distribution of myosin filaments. A similar change in
the contractile proteins may occur in hypertrophied
vascular smooth muscle from hypertensive animals
and thus account for the decreased maximal force generating ability. Bevan and Tsuru13 suggest that the diminution in force generating ability in chronically denervated arteries is due to qualitative change in the
contractile machinery.
Release and Pharmacological Displacement of
Norepinephrine from Adrenergic Nerve Endings
The release of norepinephrine in blood vessels of the
intact animal is mediated by nerve impulses.27 The
released transmitter that reaches the vascular smooth
muscle produces activation of the contractile machinery after binding to receptor sites on the cellular membranes. In this study, a variety of experimental conditions (table 1) that cause the release or displacement of
norepinephrine from nerve endings were used to characterize adrenergic neurotransmission in tail arteries
from 2K1C hypertensive and normotensive rats and
that in poorly innervated smooth muscle.
Regardless of the technique used to cause release or
displacement of norepinephrine from adrenergic nerve
endings, the maximal response of tail arteries from
2K.1C hypertensive rats which could be attributed to
endogenous norepinephrine was always less than that
in tail arteries from normotensive rats (table 1). This
response pattern was similar for chronically denervated tail arteries from normotensive rats and for arteries
with less dense adrenergic innervation. Blockade of
the neuronal uptake process did not potentiate vascular
responses to exogenous norepinephrine nor electrical
stimulation in tail arteries from hypertensive rats suggesting that the adrenergic nerve endings may have a
TABLE 1.
deficient neuronal uptake process. Vascular responses
to tyramine were less inhibited in the presence of cocaine in tail arteries from hypertensive rats than in
those from normotensive rats. Since this pharmacological displacement is dependent upon the uptake of
tyramine by the neuronal pump,27 this observation suggests that tail arteries from 2K1C hypertensive rats do
not store norepinephrine as well as arteries from normotensive rats or that there are fewer storage sites in
arteries from hypertensive rats. Furthermore, the tissue content of norepinephrine was less in tail arteries
from 2K1C hypertensive rats than in those from normotensive rats (fig. 7). Based on those observations, it
may be concluded that the adrenergic nerve endings in
blood vessels of 2K1C hypertensive rats release less
norepinephrine than do those in blood vessels from
normotensive rats. This defect is probably the result of
a reduced amount of norepinephrine available for release.
Other investigators have observed decreased adrenergic nerve function in resistance vessels of animals
with renal hypertension. Fink and Brody1 showed that
vascular responses to renal nerve stimulation and pharmacological displacement of norepinephrine by tyramine in the renal vasculature of rats with renal hypertension produced by figure 8 parenchymal ligature was
less than that in normotensive rats. Changes in vascular resistance in response to intra-arterial injection of
norepinephrine were either unchanged or greater in the
renal vascular of hypertensive rats than in those from
normotensive rats. These investigators also suggested
that diminished catecholamine storage in the kidneys
of renal hypertensive rats reflects a decreased capacity
of adrenergic nerve stimulation to produce vasoconstriction.
The current study offers no support for the hypothesis that increased sympathetic nerve activity contributes to the maintenance of renal hypertension." l2 The
results do suggest that alterations in the adrenergic
nerve ending in hypertension may serve to attenuate
vascular responsiveness to sympathetic nerve stimulation. This response of the adrenergic nerve ending may
partially explain the increased sensitivity found in this
study to norepinephrine of the vascular smooth muscle
in hypertension. It is proposed that the smooth muscle
increases its sensitivity to the catecholamine in response to the reduced release of the transmitter from
Release and Displacement of Norepinephrine
Experimental condition
Electrical stimulation
Mechanism of action
Exocytotic release
Tyramine
Pharmacological displacement
Potassium-free solution
Exocytotic release
Elevated potassium
Exocytotic release
Response of tail
artery from 2K1C
hypertensive rat
Maximal response
of chronically
denervated tail
artery
Response of aorta,
mesenteric artery.
and tail artery
Aorta < mesenteric
< tail
Aorta < mesenteric
< tail
306
HYPERTENSION
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the nerve ending. Alternatively, the vascular wall hypertrophy and/or hyperplasia (or increase in vessel
wall mass per unit length)28 that accompanies the development of hypertension results in an effective denervation of the smooth muscle cells that become increasingly farther away from their adrenergic supply at
the adventitial-medial border of the blood vessel. This
latter speculation is supported by the observation that
the vascular cells nearest the luminal surface are more
sensitive to norepinephrine whereas those nearest the
external surface are less sensitive to the catecholamine.29 w
The blood pressure of the animal may also play
some role in limiting the extent of adrenergic innervation in the blood vessel wall.29 Keatinge and Torrie29
observed that prolonged elevation of pressure throughout the vascular wall by application of a non-occluding
cylindrical clip made the entire wall of sheep carotid
arteries free of adrenergic nerves. They suggest that
the distribution of pressure within the vascular wall is
determined by two components: 1) the gradient between arterial blood pressure at the inner surface and
atmospheric pressure at the outer surface; and 2) the
relative degree of contraction of inner and outer layers
of smooth muscle. Although the importance of local
gradients in pressure are not well understood these
investigators demonstrated that the adrenergic nerves
did not survive when localized pressure within the
vascular wall approached the pressure inside the lumen
of the artery (100 mm Hg).
The observations of this study demonstrate that vascular changes in 2K1C hypertension simulate those
produced by adrenergic denervation. The precise
mechanism by which catecholamine depletion occurs
in this form of hypertension is unclear. It seems doubtful that reduced adrenergic function plays an important
role in the initiation of 2K1C hypertension. However,
the increased sensitivity of the vasculature may play a
role in the maintenance of high blood pressure when
adrenergic function is diminishing.
References
I. Fink GD, Brody MJ: Impaired neurogenic control of renal
vasculature in renal hypertensive rats. Am J Physiol 238:
H770. 1980
2 Vanhoutte PM. Webb RC. Collis MG: Pre- and post-junctional
adrenergic mechanisms and hypertension. Clin Sci 59: 211s.
1980
3. Vanhoutte PM: The adrenergic neuroeffector interaction in the
normotensive and hypertensive blood vessel wall. J Cardiovasc Pharmacol 2: (suppl 3): S-253. 1980
4. Faredin I. Benko A, Winter M. Botos A: Catecholamine content of the arterial walls in experimental hypertension. Experientia 17: 225. 1961
5. Lefer IG. Ayers CR: Norepinephrine metabolism in dogs with
chronic renovascular hypertension. Proc Soc Exp Biol Med
132: 278. 1969
6. Wegmann A. Kako K. Bing RJ: Catecholamine content of
various organs in experimental hypertension. Am J Physiol
203: 607, 1962
7. Fink GD. Brody MJ: Neurogenic control of the renal circulation in hypertension. Fed Proc 37: 1202. 1978
VOL 5, No 3, MAY-JUNE
1983
8. Ljungquist A: The role of the intrarenal sympathetic mnervation in the development of renal hypertension. Acta Pathol
Microbiol Scand 82: 450, 1974
9 Graham JDP, Lever JD, Spriggs TLB: Adrenergic innervation
of the small vessels in the pancreas of the rat and changes
observed during early renal hypertension. Circ Res 27 (suppl
2): 25, 1970
10. Azevedo I, Castro-Tavares J, Garret J: Ultrastructural changes
in blood vessels of perinephritic hypertensive dogs. Blood
Vessels 18: 110, 1981
11. Krakoff LR, DeChamplain J, Axelrod J: Abnormal storage of
norepinephrine in experimental hypertension in the rat. Circ
Res 21: 583. 1967
12. Ljungquist A, Ungerstedt U: Sympathetic innervation of juxtaglomerular cells of the kidney in rats with renal hypertension.
Acta Pathol Microbiol Scand 80: 38. 1972
13. Bevan RD, Tsuru H: Long-term denervation of vascular
smooth muscle causes not only functional but structural
change. Blood Vessels 16: 109. 1979
14. Aprigliano O. Hermsmeyer K: In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol ExpTher 198:
568, 1976
15. Webb RC. Vanhoutte PM, Bohr DF: Adrenergic neurotransmission in vascular smooth muscle from spontaneously hypertensive rats. Hypertension 3: 93, 1981
16. MahngHM. Fleisch JH, Saul WF: Species differences in aortic
responses to vasoactive amines: the effects of compound 48/
80. cocaine, reserpine and 6-hydroxydopamine. J Pharmacol
ExpTher 176: 672. 1971
17. Crabb GA. Head RJ. Hepstead J. Berkowitz BA: Altered disposition of vascular catecholamines in hypertensive (DOCAsalt) rats Clin Exp Hypertens 2: 129. 1980
18. Katholi RE. Naftilan AJ. Opanl S: Importance of renal sympathetic tone in the development of DOCA-salt hypertension in
the rat. Hypertension 2: 266. 1980
19. Vanhoutte PM. Coen EP. DeRidder WJ, Verbeuren TJ.
Evoked release of endogenous norepinephrine in the canine
saphenous vein- inhibition by acetylcholine. Circ Res 45: 608.
1979
20. Webb RC, Bohr DF. Recent advances in the pathogenesis of
hypertension: consideration of structural, functional and metabolic vascular abnormalities resulting in elevated arterial resistance. Am Heart J 102: 251, 1981
21. Bandick NR. Sparks HV: Contractile response of vascular
smooth muscle of renal hypertensive rats. Am J Physiol 219:
340, 1970
22. Hansen TR. Abrams GD, Bohr DF. Role of pressure in structural and functional changes in arteries of hypertensive rats.
Circ Res 34 (suppl I): 101. 1974
23 Hansen TR, Bohr DF: Hypertension, transmural pressure and
vascular smooth muscle response in rats. Circ Res 36: 590,
1975
24. Spector S. Fleisch JH. Mahng HM, Brodie BB: Vascular
smooth muscle reactivity in normotensive and hypertensive
rats. Science 166: 1300, 1969
25. Hansen TR. Dineen DX, Pullen GL: Orientation of arterial
smooth muscle and strength of contraction of aortic strips from
DOCA-hypertensive rats. Blood Vessels 17: 302. 1980
26. Bemer PF. Somlyo AV. Somlyo AP: Hypertrophy-induced
increase of intermediate filaments in vascular smooth muscle. J
Cell Biol 88: 96, 1981
27. Vanhoutte PM. Verbeuren TJ, Webb RC: Local modulation of
the adrenergic neuroeffector interaction in the blood vessel
wall. Physiol Rev 61: 151, 1981
28 Moreland RS, Webb RC. Bohr DF: Vascular changes in
DOCA hypertension: influence of a low protein diet. Hypertension 4 (suppl III): 111-99, 1982
29. Keatinge WR, Torrie C: Action of sympathetic nerves on inner
and outer muscle of sheep carotid artery, and effect of pressure
on nerve distribution. J Physiol 257: 699, 1976
30. de la Lande IS, Frewin D. Waterson JG: The influence of
sympathetic innervation on vascular sensitivity to noradrenaline. Br J Pharmacol 31: 82, 1967
Adrenergic neurotransmission in tail arteries from two-kidney, one clip, renal hypertensive
rats.
R C Webb, J C Johnson and D F Bohr
Hypertension. 1983;5:298-306
doi: 10.1161/01.HYP.5.3.298
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