293s
Clinical Science (1982) 63,293s-295s
Does B-endorphin contribute to the central antihypertensive
action of a-methyldopa in rats?
M . A. P E T T Y A N D W. D E J O N G
Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, The Netherlands
Abbreviation: NTS, nucleus tractus solitarii.
decrease in blood pressure and heart rate [ 1I. The
NTS is also a site of action of catecholamines and
the centrally acting hypotensive agents clonidine
and a-methyldopa 12-41. In conscious, spontaneously hypertensive rats (SHR), the decrease
in blood pressure and heart rate produced by
intravenous clonidine and a-methyldopa is reversed by naloxone [51, indicating that central
a-receptor stimulation involves the activation of
opiate receptors. Since naloxone and clonidine
appear not to interact with the same receptor site,
it was suggested that clonidine and ct-methyldopa induce the release of an endogenous opiate,
which is involved in the central control of
sympathetic tone [61. More recently it has been
reported that clonidine and a-methylnoradrenaline, the latter an active metabolite of
a-methyldopa, induce the release of a peptide
with p-endorphin-like immunoreactivity from
brain-stem slices of SHR, but not from their
normotensive, genetically matched, WistarKyoto control rats [71. This response is specific
for the active L isomer of a-methylnoradrenaline
and is reduced by yohimbine. On the basis of
these findings, it was speculated that the release
of pendorphin-like material in the brain stem
contributes to the antihypertensive action of
central cr-receptor stimulants in the SHR 171. In
this study a possible interaction between
catecholamines and endorphins has been
investigated in an experimental model of hypertension, and the NTS as a possible site of this
effect has been examined.
Introduction
Methods
PEndorphin applied locally to the nucleus tractus
solitarii (NTS) of the brain stem results in a
Male Wistar rats (Cpb, TNO, Zeist, The Netherlands) were used. Renal hypertension was induced by the application of a solid silver clip (0.2
mm internal diameter) to the left renal artery of
rats weighing 125-135 g (two kidney, one clip
Goldblatt model) 181. The control group was
Summary
1. Opioid peptide involvement in the fall in
blood pressure resulting from central a-receptor
stimulation has been investigated.
2. In conscious renal hypertensive rats (twokidney, one clip Goldblatt model), a-methyldopa
(100 mg/kg intraperitoneally) produced a longlasting fall in blood pressure which was partially
attenuated by pre-treatment with naltrexone (5
mg/kg subcutaneously).
3. Unilateral
injection
of
a-methylnoradrenaline (50 nmol) into the nucleus
tractus solitarii of normotensive, urethaneanaesthetized rats induced a fall in blood pressure
and heart rate. This fall was prevented by
pretreatment with naloxone, either 1 mg/kg
subcutaneously or 1 and 10 ng administered
directly into the nucleus.
4. Pretreatment with antiserum to Pendorphin
(1 :50 dilution), applied locally, also blocked the
depressor response induced by the catecholamine.
5. These results suggest that the fall in blood
pressure observed after a-methyldopa and its
active metabolite a-methylnoradrenaline involves
a P-endorphin-like peptide; a possible site of
action is the nucleus tractus solitarii.
Key words: /%endorphin, a-methyldopa,
methylnoradrenaline, opiates.
a-
Correspondence: Dr M. A. Petty, Rudolf Magnus
Institute for Pharmacology, Vondellaan 6, 3521 GD
Utrecht, The Netherlands.
294s
M . A . Petty and W. de Jong
subjected to the same operative procedure, but
no clip was applied. The rats were used 3-4
weeks after operation, when under light ether
anaesthesia a catheter was placed in a tail artery,
tunnelled under the skin and exteriorized at the
scruff of the neck. The animals were allowed
several hours to recover from the effects of the
anaesthesia, before drug administration and
blood pressure recordings commenced. Arterial
pressure and heart rate were recorded from the
tail artery catheter by means of a Statham
P23-AC gauge transducer and displayed on a
Grass polygraph.
For direct application of drugs into the NTS,
rats weighing 200-250 g were anaesthetized with
urethane (1.4 g/kg). The rat was placed in a
stereotactic head holder and the head fixed to an
angle of 4 5 O downward. After exposure of the
lower brain stem, unilateral micro-injections into
the NTS were given through a glass cannula
(outer diameter 60 p),in a volume of 0.4 pl,
which was delivered in 10 s. After experimentation the correct position of the injection
sites was confirmed by histology. Blood pressure
and heart rate were measured directly from a
carotid artery catheter.
The following drugs were used: (-)a-methyldopa,
(f)a-methylnoradrenaline, naloxone,
naltrexone, antiserum to Bendorphin, and control
rabbit serum. All agents were dissolved in, or
diluted with, sodium chloride solution (154
mmol/l: saline).
Results
Effect of (-)a-methyldopa in renal hypertensive
rats
The basal mean blood pressure of the conscious renal hypertensive rats ranged from 180 to
230 mmHg. a-Methyldopa (100 mg/kg intraperitoneally) induced a gradual decrease in blood
pressure, which reached a maximum at about 2.5
h after administration, when the change was -60
mmHg. An initial increase in heart rate occurred,
which persisted until about 60-90 min after
administration [31.
The opiate antagonist naltrexone (5 mg/kg
subcutaneously) did not affect either the blood
pressure or heart rate of the renal hypertensive
rats. However, when it was administered 15 min
before a-methyldopa, the decrease in blood
pressure induced by the amine was both delayed
and significantly (P < 0-05) attenuated from 2 h
onwards (Fig. 1). The changes in heart rate
induced by a-methyldopa were not affected by
naltrexone.
.I
0
1
2
3
4
Time (h)
FIG.1. Effect of naltrexone or saline pretreatment (15
min) on the fall in blood pressure induced by
a-methyldopa in conscious renal hypertensive rats:
o--a, saline + cr-methyldopa(100 mg/kg; n = 6);
L@
naltrexone (5 mg/kg) + smethyldopa (100
mg/kg) (n = 5). The results are expressed as the means
& SE, and were compared by using a Student’s t-test
for unpaired data.
Eflect of microinjection of (f)crmethylnoradrenaline into the NTS
The basal mean blood pressure and heart rate
of the urethane-anaesthetized rats ranged from 90
to 120 mmHg and 400 to 430 beatshin. Upon
administration into the NTS, a-methylnoradrenaline caused a dose-related fall in blood,
pressure and heart rate. The fall in both blood
pressure and heart rate were apparent immediately after injection. However, although the
maximum change in blood pressure occurred
after 10-15 min, the maximum decrease in heart
rate did not occur until about 25 min, both
gradually returning to control values.
Naloxone (1 mg/kg subcutaneously), administered 15 min before a-methylnoradrenaline
(50 nmol) into the NTS, attenuated the fall in
blood pressure and reduced the bradycardia
induced by the catecholamine. At the time of the
maximum change in both blood pressure and
heart rate these values were about 2 mmHg and
-20 b e a t s h i n after naloxone and -25 mmHg
and -50 b e a t s h i n after saline pretreatment
respectively.
The local application of naloxone (1-1000 ng)
into the NTS did not affect blood pressure, but 1
ng administered 10 min before a-methylnoradrenaline (50 nmol) partially antagonized the
fall in mean arterial pressure induced by the
amine, being about -30 and -10 mmHg after
saline and naloxone pretreatment respectively.
This dose of naloxone was without effect on the
Opiates and the hypotensive effects of a-methyldopa
bradycardia caused by a-methylnoradrenaline.
However, a larger dose (10 ng) of the opiate
receptor antagonist completely blocked both the
fall in blood pressure and heart rate.
Microinjection of antiserum to Pendorphin
( 1 :50 dilution) into the NTS, 10 min before
smethylnoradrenaline (50 nmol), blocked the
fall in blood pressure, being about -24 mmHg
after pretreatment with normal rabbit serum and
-5 mmHg after P-endorphin antiserum. Heart
rate decrease was reduced.
Discussion
In this study the possible involvement of an
opioid peptide in the antihypertensive effect
resulting from central @receptor activation has
been investigated. It has been reported that the
antihypertensive action of clonidine and amethyldopa in SHR is mediated by a Fendorphin-like peptide, which is released by these
centrally acting a-agonists [6, 71. Such an
interaction also appears to be present in the renal
hypertensive rat (two kidney, one clip Goldblatt
model), since the -subcutaneous administration of
naltrexone partially antagonized the fall in blood
pressure induced by a-methyldopa, injected
intraperitoneally. This interaction, however, appears to be absent in the normotensive pentobarbitone-anaesthetized rat 191, although in that
publication [91 the authors assumed that all
opioid peptides produce a blood pressure
increase.
Microinjection of a-methylnoradrenaline into
the NTS produced a rapid and marked fall in
blood pressure, which was accompanied by
bradycardia. Both the depressor response and fall
in heart rate were naloxone-reversible, the opiate
antagonist being administered subcutaneously or
directly into the NTS. These findings suggest that
the activation of a-receptors in the NTS by
a-methylnoradrenaline, to induce a fall in mean
blood pressure, requires the stimulation of opiate
receptors.
Antiserum to P-endorphin blocked the
295s
depressor response and reduced the bradycardia
resulting from a-methylnoradrenaline administration. It is possible that the catecholamine
is releasing a P-endorphin-like peptide to cause
the fall in blood pressure and heart rate.
However, it seems improbable that the opioid
peptide involved is D-endorphin, since although
&endorphin produces a decrease in blood pressure and heart rate upon microinjection into the
NTS, the time courses of these changes are much
slower than those of amethylnoradrenaline [ I].
Perhaps a shorter fragment of P L P H is involved,
with which the antiserum may cross-react.
In conclusion, it appears that a Pendorphinlike peptide is involved in the hypotensive effect
resulting from central a-receptor stimulation. The
NTS is a possible site of this action.
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