Download Is Substance P the Transmitter at the First Synapse of the

Clinical Science (1980) 5 9 , 2 9 5 ~ ~ 2 9 7 ~
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Is substance P the transmitter at the first synapse of the
baroreceptor reflex in rats and cats?
G. HAEUSLER
AND
R. O S T E R W A L D E R
Pharmaceutical Research Department of F. Hoflmann-La Roche and Co. Ltd, Basel, Switzerland
Summary
1. Substance P, injected into a lateral brain
ventricle of urethane-anaesthetized rats, caused
dose-dependent increases in blood pressure and
heart rate.
2. By contrast, exposure of the obex region of
the medulla oblongata to pieces of filter paper
soaked in substance P-containing solution
resulted in falls of blood pressure and heart rate
in both rats and cats.
3. A more precise application of substance P
to the first synapse of the baroreceptor reflex by
micro-injection into defined areas of the nucleus
of the solitary tract also led to an activation of the
baroreceptor reflex.
4. Capsaicin, which is known to release
substance P from primary afferents, mimicked
the sympatho-inhibitory and cardiovascular
effects of substance P when applied locally to the
nucleus of the solitary tract.
5. The results are compatible with a
transmitter or neuromodulatory role for
substance P at the first synapse of the baroreceptor reflex.
Key words: baroreceptor reflex, capsaicin, neuromodulator, neurotransmitter, nucleus of the
solitary tract, substance P.
Introduction
Evidence is accumulating that the undecapeptide
substance P is a neurotransmitter in the
mammalian central nervous system (for review
see Nicoll, Schenker & Leeman, 1980). The
Correspondence:
Dr
Guenther
Haeusler,
Pharmaceutical Research Department of F.
Hoffmann-La Roche and Co. Ltd, CH-4002 Basel,
Switzerland.
release of substance P has been shown to be a
calcium-dependent process (Jessel, 1978), and
electrophysiological experiments have revealed
potent excitatory effects of substance P in a
variety of regions of the central nervous system
(Henry, 1976; Guyenet & Aghajanian, 1977).
Particularly convincing are findings on pain
pathways suggesting a transmitter role of
substance P at primary sensory afferents (Henry,
1976). This has prompted us to study a potential
transmitter function of substance P in another
type of primary sensory structure, namely the
baroreceptor neurons. The transmitter released in
the nucleus of the solitary tract from the endings
of baroreceptor afferent fibres and mediating
transmission at the first synapse of the baroreceptor reflex arc has not yet been identified. The
present results are compatible with the notion
that substance P is able to excite baroreceptor
neurons in the nucleus of the solitary tract and
that it is a transmitter/neuromodulator candidate
at the first synapse of the baroreceptor reflex.
Methods
The experiments were carried out in male rats
(Wistar origin, Fullinsdorf breeding unit) and
cats (Fullinsdorf breeding unit) of either sex.
The body weights were 250-300 g and 2.5-3.1
kg respectively.
Urethane was used for anaesthesia; rats
received 1 g/kg intraperitoneally, cats 0.9 g/kg
intraperitoneally. In both species blood pressure
was recorded from the left femoral artery. The
pulse waves triggered a cardiotachometer,
allowing the meaurement of heart rate. Preganglionic and post-ganglionic sympathetic
nervous activities were measured only in cats.
The method of recording and quantification was
described previously (Haeusler, 1976).
Substance P or capsaicin was injected into a
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G . Haeusler and R . Osterwalder
lateral brain ventricle of rats after securing their
heads in a stereotaxic apparatus, and using the
following co-ordinates (Konig & Klippel, 1963):
anterior = 6.0, lateral = 1.5, vertical = 1.9.
Local application of substance P or capsaicin
to the nucleus of the solitary tract was carried
out either with the use of filter papers or as
micro-injections. For both types of application,
the occipital bone of rats or cats was removed
and free access to the obex region was obtained.
Pieces of filter paper similar in shape and size to
the rostro-caudal and medio-lateral dimensions
of the nucleus of the solitary tract, and soaked in
2 pl of substance P- or capsaicin-containing
solution, were gently placed upon the dorsal
surface of the medulla at a region corresponding
to the underlying nucleus of the solitary tract.
For micro-injection, a combined cannulaelectrode with an outer diameter of 0.3 mm and
an uninsulated tip of approximately 1 mm length
was used to explore the region of the nucleus of
the solitary tract by electrical stimulation. After
identification of sites which responded to
stimulation (8 Hz, 0.05 ms, 0.2-0.4 mA for 10
s) with a fall in blood pressure and heart rate by
at least 20 mmHg and 30 beats/min respectively,
substance P or capsaicin was micro-injected
without changing the position of the cannula
electrode.
Results
Urethane-anaesthetized rats responded to the
injection of substance P (4, 8, 16 and 24 nmol)
into a lateral brain ventricle with increases in
blood pressure and heart rate.
In contrast to the cardiovascular effects of
substance
P
after intracerebroventricular
injection, the local application of substance P to
the obex region, with the aim of obtaining a
preferential interaction of substance P with the
nucleus of the solitary tract, resulted in
pronounced falls in blood pressure, heart rate and
spontaneous sympathetic nervous activity (the
last measured only in cats). The exposure of the
obex region to small pieces of filter paper soaked
in 2 yl of a substance P solution (2 nmol/l), i.e.
containing a total amount of 4 nmol of substance
P, lowered blood pressure in rats from 102 f 3 to
85 2 3 mmHg (n = 6) and in cats from 100 & 4
to 82 k 3 mmHg (n = 6). Heart rate fell from
362 2 19 beats/min (rats) or 221 & 32 beats/
min (cats) by approximately 20%. Capsaicin
(total amount 2 or 0.2 nmol), applied in the same
way to the dorsal surface of the medulla
oblongata and tested in groups of six rats and
cats,
produced
sympatho-inhibitory
and
cardiovascular effects comparable with those
observed after substance P. Although the
cardiovascular effects of substance P applied
locally to the nucleus of the solitary tract could
be elicited six to eight times without any
decrement, only the first application of capsaicin
did so. The solvents (0.9% NaCl for substance
P, and 0- 1% or 0.01% ethanol in 0.9% NaCl for
capsaicin) were inactive.
A combined cannula-electrode with an
uninsulated tip allowed the identification of sites
within the nucleus of the solitary tract which
responded to electrical stimulation with decreases
in blood pressure and heart rate. These sites were
considered to contain the first synapse of the
baroreceptor reflex. The highest incidence of
successful stimulations was found close to the
obex in the intermediate parts of the nucleus of
the solitary tract. Subsequent micro-injection of
substance P (5, 10 and 15 nmol contained in
volumes of 2.5, 5.0 and 7.5 p l ) into these defined
areas decreased spontaneous sympathetic
nervous activity, blood pressure and heart rate of
cats in a dose-dependent manner. Similar effects
were observed in rats. Micro-injection of
capsaicin (only rat experiments) again mimicked
the effects of substance P.
Among 53 sites of the obex region where
substance P was micro-injected, virtually all
substance P-responsive sites were located in the
intermediate parts of the nucleus of the solitary
tract. Other parts of the nucleus of the solitary
tract (e.g. the rostra1 part), or those in close
vicinity but outside this nucleus, were
unresponsive.
Micro-injection into the nucleus of the solitary
tract of the solvents for substance P or capsaicin
or micro-injection of luteinizing hormonereleasing hormone, which, like substance P, is a
basic peptide of approximately similar molecular
size, failed to affect blood pressure, heart rate and
spontaneous sympathetic nervous activity.
Discussion
In contrast to the cardiovascular effects (hypertension, tachycardia) of substance P after intracerebroventricular injection, where substance P
presumably reaches a great number of brain
areas, local application of substance P to the
nucleus of the solitary tract induced decreases in
spontaneous sympathetic nervous activity, blood
pressure and heart rate. These effects are
compatible with an activation of the baroreceptor
reflex at the level of its first synapse. Non-specific
effects, possibly evoked by the technique of local
application, appear unlikely in the light of the
negative outcome of the control experiments.
Since substance P has generally been found to
be excitatory to neurons (Henry, 1976; Guyenet
Substance P and the baroreceptor reflex
& Aghajanian, 1977), the present results point to
the presence of substance P receptor sites at
baroreceptor neurons, probably at the secondorder neurons of the baroreceptor reflex. While
this does not necessarily mean that these neurons
are in fact innervated by substance P neurons,
circumstantial evidence strongly suggests the
presence of such an innervation: firstly, the
nucleus of the solitary tract is densely supplied
with substance P-containing nerve endings
(Cuello & Kanazawa, 1978; Ljungdahl, Hokfelt
& Nilsson, 1978); secondly, intracranial
sectioning of the rootlets of glossopharyngeal and
vagus nerves decreased the amount of substance
P only in the intermediate and commissural
regions of the nucleus of the solitary tract (Gillis,
Helke, Hamilton, Norman & Jacobowitz, 1980),
i.e. in those parts which contain the majority of
baroreceptor afferent nerve terminals (Miura &
Reis, 1969); thirdly, capsaicin, which is known to
release substance P from nerve endings (Gamse,
Molnar & Lembeck, 1979; Theriault, Otsuka &
Jessel, 1979), mimicked the cardiovascular effects
of substance P (present study). This last
observation seems to indicate that the substance
P-containing nerve endings are in close proximity
to the perikarya of the second-order neurons of
the baroreceptor reflex.
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References
CUELLO,A.C. & KANAZAWA,I. (1978) The distribution of
substance P immunoreactive fibers in the rat central nervous
system. Journal of Comparative Neurology, 178,129-156.
GAMSE,R., MOLNAR,A. & LEMBECK,
F. (1979) Substance P
release from spinal cord slices by capsaicin. Life Sciences, 25,
629-636.
GILLIS, R.A., HELKE,CJ., HAMILTON,
B.L., NORMAN,
W.P. &
JACOBOWITZ.
D.M. (1980) Evidence that substance P is a
neurotransmitter of baro- and chemo-receptor afferents in
nucleus tractus solitarius.Brain Research, 181,476-48 1.
GUYENET,
P.G. & AGHAJANIAN,
G.K. (1977) Excitation of neurons
in the nucleus locus coeruleus by substance P and related
peptides. Brain Research, 136,178-184.
HAEUSLER,
G. (1976) Studies on the possible contribution of a
peripheral presynaptic action of clonidine and dopamine to their
vascular effects under in vivo conditions. Naunyn-Schmiedebergk
Archives of Pharmacology, 295,19 1-202.
HENRY,J.L. (1976) Effects of substance P on functionally identified
units in cat spinal cord. Brain Research, 114,439-451.
JESSEL,T.M. (1978) Substance P release from the rat substantia
nigra. Brain Research, I5 1,469-478.
KONIG, J.F.R. & KLIPPEL,R.A. (1963) The Rat Brain. A
Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain
Stem. Williams and Wilkins, Baltimore.
LJUNODAHL,
A., HOKFELT,T. & NILSSON,G. (1978) Distribution
of substance P-like immunoreactivity in the central nervous
system of the rat. I Cell bodies and nerve terminals. Neuroscience,
3,861-943.
MIURA,M. & REIS, D J . (1969) Termination and secondary
projections of carotid sinus nerve in the cat brain stem. American
Journal of Physiology, 217,142-153.
NICOLL,
R.A., SCHENKER,
C. & LEEMAN,
S.E. (1980) Substance P
as a transmitter candidate. Annual Reviews of Neurosciences, 3,
227-268.
THERIALILT,E., OTSUKA,M. & JESSEL, T. (1979) Capsaicinevoked release of substance P from primary sensory neurons.
Brain Research, 170,209-213.