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Am J Physiol Regul Integr Comp Physiol 288: R1707–R1715, 2005.
First published February 24, 2005; doi:10.1152/ajpregu.00537.2004.
NK1 receptor activation in rat rostral ventrolateral medulla selectively
attenuates somato-sympathetic reflex while antagonism attenuates
sympathetic chemoreflex
John M. Makeham,1,2 Ann K. Goodchild,1,2 and Paul M. Pilowsky1,2
1
Hypertension and Stroke Research Laboratory, Department of Physiology, University of Sydney,
New South Wales; 2Department of Neurosurgery, Royal North Shore Hospital, Sydney, New South Wales, Australia
Submitted 9 August 2004; accepted in final form 15 February 2005
substance P; rostral ventrolateral medulla; baroreflex; brainstem; sympathetic nerve activity; neurokinin 1
THE SYMPATHOEXCITATORY CELLS of the rostral ventrolateral medulla (RVLM) project to the sympathetic preganglionic neurons of the spinal cord that are essential for the maintenance of
resting sympathetic tone (5, 21, 40, 44). The RVLM is also
essential for the integration of cardiovascular reflexes such as
the baroreflex, chemoreflex, and somato-sympathetic reflex, as
well as respiratory modulation of the sympathetic outflow (35).
Given the importance of these reflexes in cardiovascular regulation, the modulation of the sympathoexcitatory RVLM
neurons is of considerable interest. Recently, our laboratory
demonstrated that stimulation of ␦-opioid receptors in the
RVLM inhibits respiratory-related discharge of lumbar sympathetic nerve activity, while having little effect on splanchnic
sympathetic nerve activity (sSNA) and potently attenuating the
somato-sympathetic reflex. However, stimulation of ␦-opioid
Address for reprint requests and other correspondence: P. M. Pilowsky,
Hypertension and Stroke Research Laboratories, Ground Floor, Block 3, Royal
North Shore Hospital, St. Leonards, NSW 2065, Australia (E-mail:
[email protected]).
http://www.ajpregu.org
receptors in the RVLM has no effect on the baroreflex or the
chemoreflex. Stimulation of RVLM ␮-opioid receptors inhibits
the baroreflex, while leaving the somato-sympathetic reflex
and chemoreflex unaffected (28). We have also demonstrated
that activation of 5-HT1A receptors in the RVLM results in a
potent, selective inhibition of the somato-sympathetic reflex
but not the baroreflex or chemoreflex (27).
The undecapeptide tachykinin substance P and its receptor,
the neurokinin 1 (NK1) receptor, have been implicated in the
central regulation of the cardiovascular system. Microinjection
of substance P into the nucleus of the solitary tract (NTS)
modulates the baroreflex (8, 39).The RVLM contains substance P immunoreactive terminals and the NK1 receptor (12,
33). Recently, we have demonstrated that the NK1 receptor is
present on some C1 adrenergic neurons of the RVLM (24).
Furthermore, substance P immunoreactive terminals are known
to contact C1 neurons (26). Microinjection of a stable substance P analog into the RVLM causes powerful pressor
responses in vivo (43), and both substance P and the selective
NK1 receptor agonist [Sar9, Met (O2)11]SP excite neonatal
bulbospinal C1 neurons recorded using patch electrodes in
vitro (20).
In the present study, we examined the role of substance P
and the NK1 receptor in the cardiovascular functions and
reflexes mediated by the RVLM. The effects of NK1 receptor
agonist and antagonist compounds on the baroreceptor, chemoreceptor, somato-sympathetic reflex, and splanchnic sympathetic nerve activity (sSNA) were examined.
MATERIALS AND METHODS
All experimental protocols were approved by the Animal Care and
Ethics Committees of the Royal North Shore Hospital.
General procedures. Male Sprague-Dawley rats (300 –500 g) were
initially anesthetized with halothane (2% in 100% O2) followed by an
intraperitoneal injection of urethane (1.25–1.3 g/kg) and atropine (90
␮g ip). The trachea was cannulated, and the right cervical vagus nerve
was cut. The right carotid artery was catheterized for arterial blood
pressure measurement, and the right jugular vein was catheterized for
drug administration. To isolate the aortic depressor nerve (ADN) and
phrenic nerve, a longitudinal posterolateral incision was made at the
cervico-thoracic junction. After blunt dissection through the subcutaneous fat and diathermy through the parascapular muscles, the scapula
was retracted laterally, and the phrenic nerve was located posterior to
the carotid sheath at the root of the neck. The ADN was dissected
from within the carotid sheath. The splanchnic nerve was isolated
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Makeham, John M., Ann K. Goodchild, and Paul M. Pilowsky.
NK1 receptor activation in rat rostral ventrolateral medulla selectively
attenuates somato-sympathetic reflex while antagonism attenuates
sympathetic chemoreflex. Am J Physiol Regul Integr Comp Physiol
288: R1707–R1715, 2005. First published February 24, 2005;
doi:10.1152/ajpregu.00537.2004.—The effects of activation and
blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic
sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in
urethane-anesthetized and artificially ventilated Sprague-Dawley rats.
Bilateral microinjection of either the stable substance P analog
(pGlu5, MePhe8, Sar9)SP(5–11) (DiMe-SP) or the highly selective
NK1 agonist [Sar9, Met (O2)11]SP into the RVLM resulted in an
increase in ABP, sSNA, and heart rate and an abolition of phrenic
nerve activity. The effects of [Sar9, Met (O2)11]SP were blocked by
the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1
receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the
baroreflex and chemoreflex unaffected. This effect was again blocked
by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN
51708 significantly attenuated the sympathoexcitatory response to
hypoxia but had no effect on baseline respiratory function. Our
findings suggest that substance P and the NK1 receptor play a
significant role in the cardiorespiratory reflexes integrated within the
RVLM.
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NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
intermittent electrical stimulation of the tibial nerve; and 4) chemoreceptor activation by a brief period of hypoxia. Drugs were then
microinjected into the RVLM bilaterally and the activation of the
reflexes (steps 1– 4 above) was repeated. Reflex activation was repeated every 15–20 min until the return of responses to preinjection
levels. Because of significant desensitization after microinjection of
NK1 receptor agonists into the RVLM, each animal received only one
bilateral microinjection of either DiMe-SP or [Sar9, Met (O2)11]SP.
Histological procedure. At the end of each experiment, the rats
were killed with an intravenous injection of 1 M KCl. The medulla
was removed and fixed in 10% formalin in deionized water overnight.
The medulla was then sectioned transversely (100 ␮m) on a vibrating
microtome. The injection sites were identified using silver intensification of the gold particles (Sigma SE-100 Silver Enhancer Kit). The
sections were counterstained using cresyl violet and mounted on glass
slides.
Data analysis. Data were analyzed during experiments and postacquisition using a CED 1401 data capture system and Spike 4
software (Cambridge, U.K.). The average value over a 20-s period
was used to evaluate sSNA and arterial blood pressure. Phrenic
frequency and phrenic amplitude were determined using a phrenic
nerve triggered waveform average over a 100-s period. The sSNA
responses to intermittent ADN stimulation and tibial nerve stimulation
were analyzed using peristimulus waveform averaging. The amplitude
of the sSNA from ⫺200 to 0 ms before stimulation was taken as the
baseline. The maximum response to stimulation was then expressed as
a percentage change from the baseline. The response to hypoxia was
quantified by comparing the average sSNA for 10 s following the
onset of excitation of phrenic nerve discharge caused by the inhaled
100% N2 as a percent change from a control period of 10 s average
sSNA before 100% N2 inhalation.
Data are expressed as means ⫾ SE. Statistical significance was
assessed by paired t-tests to compare the effects before and after
injection of a drug. To assess the effect of treatment with DiMe-SP,
WIN 51708, and [Sar9, Met (O2)11]SP, a one-way ANOVA followed
by multiple t-tests with Bonferroni’s correction (if ANOVA were
significant) was conducted. All statistical analysis was performed
using GraphPad software.
RESULTS
Microinjection sites were located between 0 and 500 ␮m
caudal to the caudal pole of the facial nucleus, 1.9 and 2.1 mm
lateral from the midline, and ventral to the nucleus ambiguus.
A typical injection site is seen in Fig. 1.
Arterial blood pressure, sSNA, and HR. Microinjection of
DiMe-SP (600 pmol, 50 nl) bilaterally into the RVLM resulted
in a rise in arterial blood pressure (ABP) of 22 ⫾ 3 mmHg
from 96 ⫾ 6 to 119 ⫾ 5 mmHg (n ⫽ 8, P ⬍ 0.001, Fig. 3A)
Similarly, bilateral microinjection of [Sar9, Met (O2)11]SP in
the RVLM resulted in a rise in ABP of 44 ⫾ 4 mmHg from
115 ⫾ 8 to 156 ⫾ 11 mmHg (n ⫽ 6, P ⬍ 0.001, Figs. 2A and
3A). This rise was maximal at 2–5 min and lasted 30 – 40 min.
Splanchnic SNA (sSNA) and HR were also significantly
increased after microinjection of DiMe-SP and [Sar9, Met
(O2)11]SP. DiMe-SP increased sSNA to 180 ⫾ 7% baseline
(n ⫽ 7, P ⬍ 0.001, Fig. 3C) and [Sar9, Met (O2)11]SP
increased sSNA to 204 ⫾ 29% baseline (n ⫽ 6, P ⬍ 0.001,
Figs. 2A and 3C). DiMe-SP increased HR 24 ⫾ 7 bpm from
384 ⫾ 9 to 408 ⫾ 9 (n ⫽ 7, P ⬍ 0.05, Fig. 3B), whereas [Sar9,
Met (O2)11]SP increased HR 13 ⫾ 3 from 428 ⫾ 13 to 441 ⫾
15 (n ⫽ 6, P ⬍ 0.01, Fig. 3B).
Bilateral microinjection of the selective NK1 receptor antagonist WIN 51708 did not significantly change mean arterial
pressure [118 ⫾ 7 vs. 124 ⫾ 9, n ⫽ 10, not significant (NS)]
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following a longitudinal paralumbar incision and superomedially
directed blunt dissection in the retroperitoneal plane after retraction of
the paraspinal muscles and diathermy through the oblique and transverse muscle layers. Nerves were maintained in paraffin oil during
recording or stimulation. The right tibial nerve was exposed for
stimulation of the somatic afferent nerve fibers. The animals were then
secured in a stereotaxic frame, paralyzed with pancuronium dibromide
(0.8 mg iv) and artificially ventilated with O2-enriched air. End tidal
CO2 was monitored and maintained between 4 and 5% by varying the
ventilator frequency. The left cervical vagus nerve was then cut. A
partial occipital craniotomy was performed to expose the dorsal
surface of the medulla.
Adequacy of anesthesia was determined by monitoring the arterial
blood pressure and the phrenic nerve discharge. Additional doses of
urethane (20 –30 mg iv) and pancuronium dibromide (0.2 mg iv) were
given as required to maintain adequate anesthesia and neuromuscular
blockade. Rectal temperature was maintained between 36 and 38°C
with a heating pad and infrared lamp.
Nerve recording. Bipolar silver-wire electrodes were used to record
sSNA and phrenic nerve activity. The signals were amplified, filtered
(100 –3,000 Hz band pass), full-wave rectified, and integrated using a
Paynter filter with a 50-ms time constant. The zero level of sSNA was
determined using supramaximal stimulation of the aortic depressor
nerve (0.2-ms stimulation, 50 Hz for 5 s).
Activation of cardiovascular reflexes. To activate baroreceptor
afferent fibers, the ADN was stimulated electrically. Maximal activation of baroreceptor afferents was determined by tetanic ADN stimulation (0.2 ms duration, 50 Hz for 5 s). The stimulation voltage was
adjusted to achieve maximal inhibition of sSNA. This was usually
0.5– 4.0 V. To assess baroreceptor function, the average sSNA inhibition in response to intermittent ADN stimulation was determined.
The ADN was stimulated (0.2-ms duration, 2 pulses at 2.5-ms interval, 0.5 Hz), and the sSNA response was averaged at least 50 times.
Activation of the somato-sympathetic reflex was achieved by
stimulating the bipolar silver-wire cuff electrode placed around the
right tibial nerve. The nerve was stimulated at 0.5 Hz (1-ms duration,
20 –30 V).
Chemoreceptor activation was achieved by a brief period of hypoxia. Animals were ventilated with 100% N2 for 15 s.
Microinjections. The stable substance P analog [pGlu5, MePhe8,
Sar9]SP(5–11) (DiMe-SP, 600 pmol in 50 nl; Sigma), the highly
selective NK1 receptor agonist [Sar9, Met (O2)11]SP [Sar9, Met
(O2)11]SP, 600 pmol in 50 nl; Sigma) and the nonpeptide selective
NK1 receptor antagonist Win 51708 (5 nmol in 100 nl; Sigma) were
prepared for microinjection. The DiMe-SP was dissolved in 10 mM
phosphate-buffered saline (0.9%). The pH was found to be 7.4. The
[Sar9, Met (O2)11]SP was dissolved in deionized water, and the pH
was adjusted to between 7.3 and 7.5. The Win 51708 was solubilized
in 1% DMSO, and the pH was adjusted to between 7.3 and 7.5. In the
first series of experiments, multibarrel micropipettes were prepared
with either DiMe-SP or [Sar9, Met (O2)11]SP in one barrel and
albumin-colloidal gold (Sigma) in a second barrel. In the second series
of experiments, triple-barrel micropipettes were used, containing
DiMe-SP or [Sar9, Met (O2)11]SP, Win 51708 and colloidal gold in
the third barrel. The volume of injections was determined by direct
observation of the movement of the fluid meniscus in the micropipette. In all experiments the micropipettes were placed bilaterally into
the RVLM.
Experimental procedures. The pressor region of the RVLM was
identified physiologically by microinjection of L-glutamate (50 mM,
50 nl) in a single-barrel micropipette. When a site was identified
where L-glutamate microinjection elicited a pressor response of ⬎30
mmHg, the pipette was removed, and multibarrel micropipettes containing drugs were placed stereotaxically in the same sites. Reflexes
were tested in the following order: 1) baroreceptor activation by
tetanic ADN stimulation; 2) baroreceptor activation by intermittent
ADN stimulation; 3) activation of somatic afferent nerve fibers by
NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
or HR (436 ⫾ 9 vs. 442 ⫾ 10, n ⫽ 10, NS). There was a small
but significant increase in sSNA to 121 ⫾ 6% baseline (n ⫽ 10,
P ⬍ 0.01). Five to 10 min after pretreatment with WIN 51708,
either DiMe-SP or [Sar9, Met (O2)11]SP was injected into
the same site from another barrel of the triple-barrel micropipette.
Pretreatment with WIN 51708 failed to fully block the rise
in ABP following DiMe-SP, still significantly different from
baseline with an increase of 11 ⫾ 3 mmHg from 107 ⫾ 5 to
119 ⫾ 5 mmHg (n ⫽ 5, P ⬍ 0.05, Fig. 3A). However, as shown
in Figs. 2B and 3A, pretreatment with WIN 51708 blocked the
rise in ABP following [Sar9, Met (O2)11]SP (139 ⫾ 7 vs.
144 ⫾ 11 mmHg, n ⫽ 5, NS). Pretreatment with WIN 51708
blocked the HR response to DiMe-SP (from 454 ⫾ 9 to 458 ⫾
9 bpm, n ⫽ 5, NS, Fig. 3B) and to [Sar9, Met (O2)11]SP (from
438 ⫾ 18 to 433 ⫾ 17 bpm, n ⫽ 5, NS, Fig. 3B). Pretreatment
with WIN 51708 blocked the sSNA response to DiMe-SP
(114 ⫾ 6% baseline, n ⫽ 5, NS, Fig. 3C) and to [Sar9, Met
(O2)11]SP (112 ⫾ 11% baseline, n ⫽ 5, NS, Figs. 2B and 3C).
Phrenic nerve activity. Phrenic frequency was significantly
reduced after bilateral DiMe-SP microinjection to 39 ⫾ 7%
baseline (n ⫽ 8, P ⬍ 0.001, Fig. 3D). Phrenic nerve activity
was consistently abolished following [Sar9, Met (O2)11]SP
microinjection. This inhibition lasted 2–5 min (Fig. 2A). There
was no significant decrease in phrenic nerve amplitude after
DiMe-SP (102 ⫾ 5% baseline, n ⫽ 8) or upon return of phrenic
nerve activity after [Sar9, Met (O2)11]SP (104 ⫾ 9% baseline,
n ⫽ 6). Bilateral microinjection of WIN 51708 did not significantly alter phrenic frequency (30 ⫾ 2 vs. 31 ⫾ 3/min, n ⫽ 10,
NS) or phrenic amplitude (102 ⫾ 6% baseline, n ⫽ 10, NS).
Pretreatment with WIN 51708 failed to block the phrenic
frequency response to DiMe-SP, reduced to 51 ⫾ 11% baseline
(34 ⫾ 4 vs. 19 ⫾ 6/min, n ⫽ 5, P ⬍ 0.05, Fig. 3D) but did
block the phrenic frequency response to [Sar9, Met (O2)11]SP
(32 ⫾ 5 vs. 22 ⫾ 9/min, n ⫽ 5, NS, Fig. 3D).
Somato-sympathetic reflex. The average SNA response to
intermittent stimulation of the tibial nerve was tested both
before and after microinjection of drugs. Intermittent tibial
nerve stimulation resulted in a characteristic two-peaked response in the sSNA with the latencies of 115 ⫾ 2 ms and
211 ⫾ 4 ms (n ⫽ 7, Fig. 4A). Bilateral microinjection of
DiMe-SP in the RVLM significantly attenuated the first and
second peaks to 27 ⫾ 10% and 1 ⫾ 8% baseline, respectively
(n ⫽ 6, P ⬍ 0.001). Bilateral microinjection of [Sar9, Met
(O2)11]SP significantly attenuated the first peak to 15 ⫾ 3%
baseline (n ⫽ 5, P ⬍ 0.05, Fig. 4, A and C). The second peak
of the somato-sympathetic reflex was often absent during
baseline stimulation in these experiments. On two occasions, it
was shown to be attenuated to 8% and 9% baseline by [Sar9,
Met (O2)11]SP microinjection (Fig. 4C).
Bilateral microinjection of WIN 51708 did not significantly
attenuate either the first or second peak of the somato-sympathetic reflex (83 ⫾ 30%; n ⫽ 10 and 64 ⫾ 19%; n ⫽ 9 baseline,
respectively, NS). Pretreatment with WIN 51708 failed to
block the response to DiMe-SP of the first peak, still attenuated
to 58 ⫾ 11% baseline (n ⫽ 5, P ⬍ 0.05), but the response of
the second peak was blocked (82 ⫾ 31% baseline, n ⫽ 4, NS).
Pretreatment with WIN 51708 blocked the attenuation of the
somato-sympathetic response caused by [Sar9, Met (O2)11]SP
for both the first and second peaks (104 ⫾ 43% baseline and
172 ⫾ 56% baseline, respectively; n ⫽ 5, NS, Fig. 4B and C).
Baroreflex. Intermittent stimulation of the ADN resulted in a
maximal inhibitory potential in the sSNA with a latency of
178 ⫾ 4 ms (n ⫽ 13, Fig. 5C). Bilateral microinjection of
DiMe-SP attenuated the amplitude of the inhibitory potential
58 ⫾ 9% (n ⫽ 7, P ⬍ 0.01), whereas [Sar9, Met (O2)11]SP did
not significantly attenuate the inhibitory potential (76 ⫾ 11%
baseline, n ⫽ 5, NS, Fig. 5C). Bilateral microinjection of WIN
51708 did not significantly alter the response to intermittent
ADN stimulation (90 ⫾ 8% baseline, n ⫽ 10, NS). Pretreatment with WIN 51708 blocked the response to both DiMe-SP
(89 ⫾ 5% baseline, n ⫽ 5, NS) and [Sar9, Met (O2)11]SP
(109 ⫾ 17% baseline, n ⫽ 5, NS). An example is shown in
Fig. 5C.
Chemoreflex. Stimulation of the chemoreflex resulted in a
characteristic increase in sSNA from prestimulus levels of
170 ⫾ 11% (n ⫽ 9, P ⬍ 0.01, Fig. 5A). Expressed as a
percentage of the baseline response, microinjection of either
DiMe-SP or [Sar9, Met(O2)11]SP failed to significantly alter
the chemoreflex (80 ⫾ 13%; n ⫽ 7, NS and 90 ⫾ 24%; n ⫽ 6,
NS) baseline, respectively. Bilateral microinjection of WIN
51708, however, resulted in a significant attenuation of the
chemoreflex, to 38 ⫾ 5% baseline (n ⫽ 9, P ⬍ 0.001, Fig. 5,
A and B). The interval between baseline chemoreflex testing
and chemoreflex testing with the NK1 receptor agonist drugs,
either DiMe-SP or [Sar9, Met (O2)11]SP, was not significantly
different from the interval between baseline chemoreflex testing and that after pretreatment with the NK1 receptor antagonist WIN 51708: 986 ⫾ 63 s (n ⫽ 13) vs. 1116 ⫾ 113 s (n ⫽
9), NS.
DISCUSSION
The principal novel findings of this study are 1) activation of
NK1 receptors in the RVLM in vivo results in hypertension,
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Fig. 1. Microinjection site in the rostral ventrolateral medulla (RVLM). The
arrowhead identifies the microinjection site marked with silver-intensified gold
particles. Injections into the RVLM were located caudal to the caudal pole of
the facial nucleus and ventral to the compact formation of the nucleus
ambiguus. D, dorsal; M, midline. Scale bar ⫽ 500 ␮m.
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NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
tachycardia, and splanchnic sympathoexcitation; 2) activation
of NK1 receptors in the RVLM attenuates the somato-sympathetic reflex without affecting other brainstem reflexes such as
the baroreflex or chemoreflex, and; 3) blockade of NK1 receptors in the RVLM significantly attenuates the sSNA response to
chemoreceptor stimulation. Activation of NK1 receptors in the
RVLM was also shown to significantly decrease phrenic frequency.
Activation of NK1 receptors in the RVLM in vivo with
DiMe-SP has previously been shown to increase ABP and HR
(43). Our results agree with these findings. Further, the present
study demonstrates a significant increase in sSNA following
bilateral NK1 receptor stimulation in the RVLM. This is most
likely mediated, in part, by NK1 receptors found on bulbospinal C1 neurons, which are sympathoexcitatory (24). Substance
P terminals have been demonstrated within the RVLM and
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Fig. 2. A: bilateral microinjection of [Sar9,
Met (O2)11]SP into the RVLM (solid arrowheads) elicited a sympathoexcitatory response and a rise in arterial blood pressure
(ABP). There was also a complete inhibition
of phrenic nerve activity, returning in ⬃400
s. Open arrows indicate tetanic stimulation of
the aortic depressor nerve (ADN). Asterisk
indicates ventilation with 100% N2 for 15 s.
During the sympathoexcitatory period, multiple reflexes were tested, including the baroreflex, somato-sympathetic reflex, and chemoreflex. sSNA, splanchnic sympathetic
nerve activity. B: bilateral microinjection of
[Sar9, Met (O2)11]SP after pretreatment with
WIN 51708 in the RVLM. WIN 51708 had
no significant effect on sympathetic or
phrenic nerve activity but did cause a small
but significant rise in ABP (left). After pretreatment with WIN 51708, [Sar9, Met
(O2)11]SP had no significant effect on arterial blood pressure, sympathetic nerve activity, or phrenic nerve activity (right).
form synaptic junctions with C1 neurons (18, 26). Substance P
and [Sar9, Met (O2)11]SP excite neonatal bulbospinal C1
neurons in vitro, possibly by a reduction in resting potassium
conductance (20).
Pretreatment of the RVLM with the highly selective NK1
receptor antagonist WIN 51708 attenuated the pressor response
to DiMe-SP and blocked the effects of [Sar9, Met (O2)11]SP.
This suggests that the pressor response to DiMe-SP may be
partially mediated by binding sites that are different to the
binding sites activated by [Sar9, Met (O2)11]SP. The carboxyterminal substance P analogs such as DiMe-SP may preferentially activate NK3 receptors (9). NK3 receptors are present
within the reticular formation of the medulla (25). Senktide, an
NK3 agonist, excited a small number (two out of seven
neurons) of neonatal bulbospinal C1 neurons recorded using in
vitro patch clamp (20).
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NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
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Pretreatment of the RVLM with WIN 51708 blocked the
sympathoexcitatory effects of both DiMe-SP and [Sar9, Met
(O2)11]SP. There was a small but significant increase in sSNA
immediately after administration of WIN 51708. The exact
cause of this unknown, but it may be due to a partial agonist
activity of WIN 51708, as has been demonstrated previously in
other tachykinin antagonists (16, 37).
Bilateral microinjection of both DiMe-SP and [Sar9, Met
(O2)11]SP into the RVLM markedly attenuated and completely
abolished phrenic nerve activity, respectively. Immediately
dorsal to the sympathoexcitatory neurons of the RVLM are the
Bötzinger neurons of the ventral respiratory group (36, 42).
Microinjection of excitatory amino acids in the Bötzinger
complex decreases phrenic nerve burst amplitude and frequency (3, 4, 7, 45). This is presumably due to activation of
inhibitory expiratory neurons (10, 38). NK1 receptors have
also been demonstrated on both spinally and nonspinally projecting neurons within the Bötzinger/RVLM region (11, 24).
Activation of NK1 receptors in the Bötzinger region may thus
activate glycinergic inhibitory expiratory neurons resulting in
an inhibition of phrenic nerve activity. As previous studies
have suggested that the majority of NK1 receptor immunoreactive neurons of the ventral respiratory group are glutamatergic (11), the activation of inhibitory expiratory neurons may
alternatively occur through an excitatory NK1 receptor immunoreactive interneuron. The fact that NK1 receptor activation
within this region resulted in a robust inhibition of phrenic
nerve activity, whereas NK1 receptor blockade had no effect,
suggests that endogenous NK1 receptor agonists are not constitutively active in the respiratory-related neurons in this
region. Activation of NK1 receptors on respiratory-related
neurons in the RVLM may occur only under stressful physiological or pathological states. Further studies are required to
uncover the mechanism of phrenic nerve activity inhibition by
NK1 receptor activation in the Bötzinger region.
Electrical stimulation of hindlimb somatic afferent nerves
evokes an early and late excitatory response in SNA (27, 31).
The excitatory peaks found in this experiment were similar in
morphology and latency to those described in previous work
from our laboratory and elsewhere (23, 27, 28, 32, 46). This
reflex is dramatically attenuated by blockade of, for example,
excitatory amino acid receptors in the RVLM (13, 29), and by
activation of 5-HT1A (27) and ␦-opioid receptors in the RVLM
(28), and by hypercapnia (23).
Activation of NK1 receptors in the RVLM by [Sar9, Met
(O2)11]SP abolished both peaks of the somato-sympathetic
reflex, an effect blocked by WIN 51708. Given that activation
of NK1 receptors in the RVLM produces a large sympathoexcitatory response, while leaving the baroreflex unaffected, it is
unlikely that NK1 receptor activation abolishes the somatosympathetic reflex via a direct effect on bulbospinal C1 neurons. It may be that NK1 receptor immunoreactive terminals
within the RVLM contain inhibitory neurotransmitters such as
GABA or glycine and inhibit or selectively gate excitatory
somatic inputs. We have previously demonstrated that NK1
receptor immunoreactive terminals exist in the RVLM, and
some make close apposition with C1 neurons (24).
It is important to note that two forms of the NK1 receptor
exist: a long form and a C-terminus-truncated form (19). The
commercially available NK1 receptor antibody only recognizes
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Fig. 3. Group data for the effects of
DiMe-SP (DiMe), [Sar9, Met (O2)11]SP
(SarMet-SP) and WIN 51708 (WIN) within
the RVLM on changes in ABP (A), changes
in heart rate (HR; B), splanchnic sympathetic
nerve activity (sSNA; C) and phrenic frequency (D). Note the increase in ABP, HR,
and sSNA and the decrease in phrenic frequency after neurokinin 1 receptor (NK1R)
activation with either DiMe or SarMet-SP.
The highly selective NK1R antagonist WIN
blocked the effects of SarMet-SP but only
partially attenuated the effects of DiMe on
ABP and phrenic frequency. ⫹P ⬍ 0.05 vs.
baseline. *P ⬍ 0.01 vs. baseline. **P ⬍
0.001 vs. baseline.
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NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
the long form, and by definition, anatomical studies using this
antibody do not demonstrate immunoreactivity to the short
form. In humans and guinea pigs, up to 30% of the NK1
receptor population within the medulla oblongata is of the short
form (2, 6). Further studies are required to demonstrate the
distribution of the short form of the NK1 receptor within the
ventral medulla.
Microinjection of a 5-HT1A agonist into the RVLM markedly attenuates the somato-sympathetic reflex while leaving the
baroreflex unaffected (27), a result similar to the finding in this
study. This indicates the possibility that inhibition of the
somato-sympathetic reflex by NK1 receptor activation may
occur through a pathway involving 5-HT1A receptors in the
RVLM. However, there is evidence that the major nuclei
within the brainstem that have serotonergic projections to the
RVLM do not display dual labeling for 5-HT and the NK1
receptor (1, 17). Nevertheless, it is possible that within the
RVLM, substance P causes activation of presynaptic NK1
receptors on excitatory inputs to serotonergic projections, re-
sulting in 5-HT release and subsequent binding to 5-HT1A
receptors. This mechanism has previously been demonstrated
in the dorsal raphe nucleus in the rat (22). As mentioned
previously, it is also possible that serotonergic neurons might
express the short form of the NK1 receptor (1, 17).
The possibility that the significant increase in sSNA following NK1 receptor activation might itself attenuate the expression of both peaks of the somato-sympathetic reflex must also
be considered. This is unlikely to be the case for two reasons.
First, the increase in sSNA, though large, is not at the level
where no further increase is possible. Following glutamate
microinjection into the RVLM, maximum sSNA was found to
be 2–3 times as great as the level attained by NK1 receptor
stimulation (data not shown). Second, RVLM NK1 receptor
activation did not alter the sympathetic baroreflex, a reflex
mediated by the same neurons.
An interesting finding is the effect of the highly selective
NK1 receptor antagonist WIN 51708 on the sSNA chemoreflex
response. The consistency of the hypoxic stimulus must be
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Fig. 4. A: electrical stimulation of the tibial nerve
(solid arrowheads) produced the two characteristic
peaks of the somato-sympathetic reflex in the sSNA
recording. Bilateral microinjection of [Sar9, Met
(O2)11]SP in the RVLM almost completely abolished this response. This abolition lasted ⬃60 –90
min, followed by partial recovery. B: RVLM pretreatment with WIN 51708 blocked the effects of
[Sar9, Met (O2)11]SP on the somato-sympathetic
reflex. C: group data for the first and second peaks of
the somato-sympathetic reflex. Note the almost complete inhibition of peak 1 and peak 2 after [Sar9, Met
(O2)11]SP and the blocking of this effect by WIN
51708. Note also that peak 2 was absent in many
experiments, and the effect of [Sar9, Met (O2)11]SP
is only measured in two animals; however, in these
animals the inhibition of peak 2 was to 8% and 9%
baseline. *P ⬍ 0.05 vs. baseline. **P ⬍ 0.001 vs.
baseline.
NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
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considered. Experiments were conducted in a standardized
manner, and animals were ventilated so as to maintain end tidal
CO2 between 4 and 5%. Sympathoexcitation occurred rapidly
after onset of ventilation with 100% N2, and the measured
chemoreflex period occurred wholly within the 100% N2 administration period. Furthermore, the measured chemoreflex
period was an average of 10 s immediately following the onset
of excitation of phrenic nerve discharge. These factors suggest
a consistent hypoxic stimulus.
Hypoxia stimulates carotid body chemoreceptors and increases the activity of bulbospinal sympathoexcitatory neurons
in the RVLM via the NTS (14, 30, 41). Blockade of excitatory
amino acid neurotransmitters in the RVLM also blocks the
sympathoexcitation evoked by the chemoreflex (14, 15, 30,
41). There is some evidence that NK1 receptor-containing
neurons of the ventral medulla may be chemosensitive or
modulate chemosensitivity (34). However, the fact that antagonism of NK1 receptors in the RVLM did not alter baseline
respiratory frequency or phrenic nerve amplitude suggests that
within this region the NK1 receptor is not involved in basal
respiratory function. The NK1 receptor appears to play a role
in sympathoexcitation within the RVLM under stressful stimuli, such as the severe hypoxia experienced within this experimental protocol. Substance P may be presynaptically reinforcing the excitatory NTS to RVLM connection (14, 30, 41).
Removing this by NK1 receptor blockade would thus reduce
the sympathetic response. The apparent failure of either
DiMe-SP or [Sar9, Met (O2)11]SP to augment the chemoreflex
may be due to the fact that the chemoreceptor facilitation is
masked by the general sympathoexcitation evoked by the
RVLM NK1 receptor activation.
In summary, we find that in anesthetized, vagotomized,
paralyzed, and ventilated rats, microinjection of NK1 receptor
agonists bilaterally into the RVLM elicits robust increases in
ABP, sSNA, and HR. It also results in the abolition of phrenic
nerve activity and dramatic attenuation of the somato-sympathetic reflex while leaving the baroreflex unaffected. These
effects are due to the selective activation of the NK1 receptor
within the RVLM, as evidenced by the blocking of these
effects by a highly selective NK1 receptor antagonist. The fact
that the selective NK1 receptor antagonist had no significant
effect on baseline ABP and HR and a very small effect on
sSNA suggests that substance P and the NK1 receptor do not
play a significant role in the tonic maintenance of blood
pressure and sympathetic outflow. NK1 receptor antagonism
also resulted in a dramatic attenuation of the sympathetic
chemoreflex response to hypoxia, suggesting a modulation of
the chemoreceptor pathway from the NTS to the RVLM by
substance P. These results demonstrate that substance P and the
NK1 receptor play an integral role in the regulation of cardiorespiratory reflexes within the RVLM.
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Fig. 5. A: 15 s of 100% N2 resulted in robust
excitation and rise in ABP (left). This response was not affected by either DiMe-SP or
[Sar9, Met (O2)11]SP but was significantly
attenuated by bilateral RVLM microinjection
of WIN 51708 (right). B: group data for the
effects of DiMe-SP, [Sar9, Met (O2)11]SP,
and WIN 51708 on the chemoreflex. **P ⬍
0.001 vs. baseline. C: intermittent stimulation
of the aortic depressor nerve (solid arrowheads) resulted in a characteristic inhibitory
trough in sSNA with a latency of 178 ⫾ 4 ms.
Microinjection of DiMe-SP resulted in attenuation of this inhibition, whereas the highly
specific NK1 receptor agonist [Sar9, Met
(O2)11]SP and antagonist WIN 51708 had no
significant effect.
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NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
GRANTS
The study was supported by grants from the National Health and Medical
Research Council of Australia, the National Heart Foundation, The North
Shore Heart Research Foundation, the Doug White AVM Foundation, the
Andrew Olle Memorial Trust, the Garnett Passe and Rodney Williams Memorial Foundation, and the High Blood Pressure Foundation of Australia.
REFERENCES
AJP-Regul Integr Comp Physiol • VOL
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1. Bago M, Marson L, and Dean C. Serotonergic projections to the
rostroventrolateral medulla from midbrain and raphe nuclei. Brain Res
945: 249 –258, 2002.
2. Baker SJ, Morris JL, and Gibbins IL. Cloning of a C-terminally
truncated NK-1 receptor from guinea-pig nervous system. Mol Brain Res
111: 136 –147, 2003.
3. Bongianni F, Fontana G, and Pantaleo T. Effects of electrical and
chemical stimulation of the Bötzinger complex on respiratory activity in
the cat. Brain Res 445: 254 –261, 1988.
4. Bongianni F, Mutolo D, and Pantaleo T. Depressant effects on inspiratory and expiratory activity produced by chemical activation of Bötzinger
complex neurons in the rabbit. Brain Res 749: 1–9, 1997.
5. Brown DL and Guyenet PG. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ Res 56:
359 –369, 1985.
6. Caberlotto L, Hurd YL, Murdock P, Wahlin JP, Melotto S, Corsi M,
and Carletti R. Neurokinin 1 receptor and relative abundance of the short
and long isoforms in the human brain. Eur J Neurosci 17: 1736 –1746,
2003.
7. Chitravanshi VC and Sapru H. GABA receptors in the phrenic nucleus
of the rat. Am J Physiol Regul Integr Comp Physiol 276: R420 –R428,
1999.
8. Cowan AR, Dean C, Bago M, and Seagard JL. Potentiation of non-Nmethyl-D-aspartate receptor-induced changes in blood pressure by substance P in rats. Neurosci Lett 278: 161–164, 2000.
9. Drapeau G, Dorleansjuste P, Dion S, Rhaleb NE, Rouissi NE, and
Regoli D. Selective agonists for substance-P and neurokinin receptors.
Neuropeptides 10: 43–54, 1987.
10. Duffin J, Ezure K, and Lipski J. Breathing rhythm generation: Focus on
the rostral ventrolateral medulla. News Physiol Sci 10: 133–140, 1995.
11. Guyenet PG, Sevigny CP, Weston MC, and Stornetta RL. Neurokinin-1 receptor-expressing cells of the ventral respiratory group are functionally heterogeneous and predominantly glutamatergic. J Neurosci 22:
3806 –3816, 2002.
12. Helke CJ, Shults CW, Chase TN, and O’Donohue TL. Autoradiographic localization of substance P receptors in rat medulla: effect of
vagotomy and nodose ganglionectomy. Neuroscience 12: 215–223, 1984.
13. Kiely JM and Gordon FJ. Non-NMDA receptors in the rostral ventrolateral medulla mediate somatosympathetic pressor responses. J Auton
Nerv Syst 43: 231–240, 1993.
14. Koshiya N, Huangfu D, and Guyenet PG. Ventrolateral medulla and
sympathetic chemoreflex in the rat. Brain Res 609: 174 –184, 1993.
15. Kubo T, Amano M, and Asari T. N-methyl-D-aspartate receptors but not
non-N-methyl-D-aspartate receptors mediate hypertension induced by carotid body chemoreceptor stimulation in the rostral ventrolateral medulla
of the rat. Neurosci Lett 164: 113–116, 1993.
16. Kudlacz EM, Logan DE, Shatzer SA, Farrell AM, and Baugh LE.
Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists. Eur J Pharmacol 241:
17–25, 1993.
17. Leger L, Gay N, and Cespuglio R. Neurokinin NK1- and NK3-immunoreactive neurons in serotonergic cell groups in the rat brain. Neurosci
Lett 323: 146 –150, 2002.
18. Leibstein AG, Dermietzel R, Willenberg IM, and Pauschert R. Mapping the different neuropeptides in the lower brainstem of the rat: with
special reference to the ventral surface. J Auton Nerv Syst 14: 299 –313,
1985.
19. Li H, Leeman SE, Slack BE, Hauser G, Saltsman WS, Krause JE,
Krzysztof Blusztajn J, and Boyd ND. A substance P (neurokinin-1)
receptor mutant carboxyl-terminally truncated to resemble a naturally
occurring receptor isoform displays enhanced responsiveness and resistance to desenstization. Proc Natl Acad Sci USA 94: 9475–9480,
1997.
20. Li YW and Guyenet PG. Effect of substance P on Cl and other
bulbospinal cells of the RVLM in neonatal rats. Am J Physiol Regul Integr
Comp Physiol 273: R805–R813, 1997.
21. Lipski J, Kanjhan R, Kruszewska B, and Smith ML. Barosensitive
neurons in the rostral ventrolateral medulla of the rat in vivo: morphological properties and relationship to C1 adrenergic neurons. Neuroscience
69: 601– 618, 1995.
22. Liu RL, Ding Y, and Aghajanian GK. Neurokinins activate local
glutamatergic inputs to serotonergic neurons of the dorsal raphe nucleus.
Neuropsychopharmacology 27: 329 –340, 2002.
23. Makeham JM, Goodchild AK, Costin NS, and Pilowsky PM. Hypercapnia selectively attenuates the somato-sympathetic reflex. Respir
Physiol Neurobiol 140: 133–143, 2004.
24. Makeham JM, Goodchild AK, and Pilowsky PM. NK1 receptor and the
ventral medulla of the rat: bulbospinal and catecholaminergic neurons.
Neuroreport 12: 3663–3667, 2001.
25. Mileusnic D, Lee JM, Magnuson DJ, Hejna MJ, Krause JE, Lorens
JB, and Lorens SA. Neurokinin-3 receptor distribution in rat and human
brain: an immunohistochemical study. Neuroscience 89: 1269 –1290,
1999.
26. Milner TA, Pickel VM, Abate C, Joh TH, and Reis DJ. Ultrastructural
characterization of substance P-like immunoreactive neurons in the rostral
ventrolateral medulla in relation to neurons containing catecholaminesynthesizing enzymes. J Comp Neurol 270: 427– 445, 1988.
27. Miyawaki T, Goodchild AK, and Pilowsky PM. Rostral ventral medulla
5-HT1A receptors selectively inhibit the somatosympathetic reflex. Am J
Physiol Regul Integr Comp Physiol 280: R1261–R1268, 2001.
28. Miyawaki T, Goodchild AK, and Pilowsky PM. Activation of Muopioid receptors in rat ventrolateral medulla selectively blocks baroreceptor reflexes while activation of delta opioid receptors blocks somatosympathetic reflexes. Neuroscience 109: 133–144, 2002.
29. Miyawaki T, Minson JB, Arnolda LF, Chalmers JP, Llewellyn-Smith
IJ, and Pilowsky PM. Role of excitatory amino acid receptors in cardiorespiratory coupling in the ventrolateral medulla. Am J Physiol Regul
Integr Comp Physiol 271: R1221–R1230, 1996.
30. Miyawaki T, Minson JB, Arnolda LF, Llewellyn-Smith IJ, Chalmers
JP, and Pilowsky PM. AMPA/kainate receptors mediate sympathetic
chemoreceptor reflex in the rostral ventrolateral medulla. Brain Res 726:
64 – 68, 1996.
31. Morrison SF and Reis DJ. Reticulospinal vasomotor neurons in the RVL
mediate the somatosympathetic reflex. Am J Physiol Regul Integr Comp
Physiol 256: R1084 –R1097, 1989.
32. Nagata O, Li WM, and Sato A. Glutamate N-methyl-D-aspartate
(NMDA) and non-NMDA receptor antagonists administered into the brain
stem depress the renal sympathetic reflex discharges evoked by single
shock of somatic afferents in anesthetized rats. Neurosci Lett 201: 111–
114, 1995.
33. Nakaya Y, Kaneko T, Shigemoto R, Nakanishi S, and Mizuno N.
Immunohistochemical localization of substance P receptor in the central
nervous sstem of the adult rat. J Comp Neurol 347: 249 –274, 1994.
34. Nattie EE and Li A. Substance P-saporin lesion of neurons with NK1
receptors in one chemoreceptor site in rats decreases ventilation and
chemosensitivity. J Physiol 44: 603– 616, 2002.
35. Pilowsky PM and Goodchild AK. Baroreceptor reflex pathways and
neurotransmitters: 10 years on. J Hypertens 20: 1675–1688, 2002.
36. Pilowsky PM, Jiang C, and Lipski J. An intracellular study of respiratory neurons in the rostral ventrolateral medulla of the rat and their
relationship to catecholamine-containing neurons. J Comp Neurol 301:
604 – 617, 1990.
37. Schreihofer AM, Stornetta RL, and Guyenet PG. Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycinergic transporter 2. J Comp Neurol 407: 583–597, 1999.
38. Sachon E, Girault-Lagrange S, Chassaing G, Lavielle S, and Sagan S.
Analogs of Substance P modified at the C-terminus which are both agonist
and antagonist of the NK-1 receptor depending on the second messenger
pathway. J Pept Res 59: 232–240, 2002.
39. Seagard JL, Dean C, and Hopp FA. Modulation of the carotid baroreceptor reflex by substance P in the nucleus tractus solitarius. J Auton Nerv
Syst 78: 77– 85, 2000.
40. Sun MK. Central neural organization and control of sympathetic nervous
system in mammals. Prog Neurobiol 47: 157–233, 1995.
NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM
41. Sun MK and Reis DJ. NMDA receptor-mediated sympathetic chemoreflex excitation of RVL-spinal vasomotor neurons in rats. J Physiol 482:
53– 68, 1995.
42. Sun QJ, Minson JB, Llewellyn-Smith IJ, Arnolda LF, Chalmers JP,
and Pilowsky PM. Bötzinger neurons project toward bulbospinal neurons
in the rostral ventrolateral medulla of the rat. J Comp Neurol 388: 23–31,
1997.
43. Urbanski RW, Murugaian J, Krieger AJ, and Sapru HN. Cardiovascular effects of substance P receptor stimulation in the ventrolateral
medullary pressor and depressor areas. Brain Res 491: 383–389, 1989.
R1715
44. Verberne AJM, Stornetta RL, and Guyenet PG. Properties of C1 and
other ventrolateral medullary neurons with hypothalamic projections in the
rat. J Physiol 517: 477– 494, 1999.
45. Wang H, Germanson TP, and Guyenet PG. Depressor and tachypneic
responses to chemical stimulation of the ventral respiratory group are
reduced by ablation of neurokinin-1 receptor-expressing neurons. J Neurosci 22: 3755–3764, 2002.
46. Zanzinger J, Czachurski J, Offner B, and Seller H. Somato-sympathetic reflex transmission in the ventrolateral medulla oblongata: spatial
organization and receptor types. Brain Res 656: 353–358, 1994.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
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