Attenuation of reflex pressor and ventilatory responses to static contraction by an NK-I receptor antagonist JANEEN M. HILL, JOEL G. PICKAR, AND MARC P. KAUFMAN Division of Cardiovascular Medicine, Departments of Internal Medicine and Human Physiology, University of California, Davis, California 95616 HILL,JANEEN M., JOELG.PICKAR,ANDMARC P. KAUFMAN. Attenuation of reflex pressor and ventilatory responses to static contraction by an NK-1 receptor antagonist. J. Appl. Physiol. 73(4): 1389-1395, 1992.-The chemical messengers released onto second-order dorsal horn neurons from the spinal terminals of contraction-activated group III and IV muscle afferents have not been identified. One candidate is the tachykinin substance P. Related to substance P are two other tachykinins, neurokinin A (NKA) and neurokinin B (NKB), which, like substance P, have been isolated in the dorsal horn of the spinal cord and have receptors there. Whether NKA or NKB plays a transmitter/modulator role in the spinal processing of the exercise pressor reflex is unknown. Therefore, we tested the following hypotheses. After the intrathecal injection of a highly selective NK-1 (substance P) receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Likewise, after the intrathecal injection either of an NK-2 (NKA) receptor antagonist or an NK-3 (NKB) receptor antagonist onto the lumbrosacral spinal cord, the reflex pressor and ventilatory responses to static contraction will be attenuated. We found that, 10 min after the intrathecal injection of 100 pg of the NK-1 receptor antagonist, the pressor and ventilatory responses to contraction were significantly (P < 0.05) attenuated. Mean arterial pressure was attenuated by 13 * 3 mmHg (48%) and minute volume of ventilation by 120 t 38 ml/min (34%). The cardiovascular and ventilatory responses to contraction before either 100 pg of the NK-2 receptor antagonist or 100 pg of the NK-3 receptor antagonist were not different (P > 0.05) from those after the NK-2 or the NK-3 receptor antagonists. We were unable to demonstrate a role for NK-2 (NKA) and NK-3 (NKB) receptors in the spinal processing of the exercise pressor reflex. Our results provide evidence that substance P, acting via the NK-1 receptor, is involved in the transmission of contraction-activated afferent input to the dorsal horn of the spinal cord. exercise; cats; tachykinins; circulation; peptides
substance
P; CP 96,345; control
of
THEEXERCISEPRESSORREFLEX referstothe reflexcardiovascular and ventilatory increases evoked by static muscular contraction (19). The afferent arm of this reflex arc is comprised of group III and IV fibers, which are innervated by sensory endings in the contracting muscle and which synapse in the dorsal horn of the spinal cord (18). The efferent arm of this reflex arc is comprised of the sympathetic outflow to the heart and blood vessels, the parasympathetic nerves to the heart, and the motor nerves to the respiratory muscles. 0161-7567/92 $2.00 Copyright
The chemical messengers released onto the secondorder dorsal horn neurons from the spinal terminals of contraction-activated group III and IV fibers have not been determined. One important candidate is the tachykinin substance P (SP), which has a role as a spinal neurotransmitter/modulator in the exercise pressor reflex that has been suggested by the findings of several studies. For example, SP has been found in the spinal terminals of fine-caliber axons running in the L,--S, dorsal roots (10). SP has been shown to be released in the spinal cord when A& and C-fibers in the sciatic nerve have been electrically stimulated (29). Iontophoresis of SP has been shown to stimulate dorsal horn cells receiving somatic afferent input (6). Finally, SP receptors [neurokinin-1 (NK-l)] have been identified immunohistochemically in the dorsal horn (21). Three previous studies have demonstrated an attenuated pressor response to static muscular contraction after the microinjection of a SP antagonist into the L, dorsal horn (28) or after the intrathecal injection of a SP antagonist (12) or antibody (13) onto the feline lumbosacral spinal cord. Since these findings, the antagonist used in both the microinjection and intrathecal studies has been shown to exhibit an affinity for the neurokinin A (NKA) receptor NK-2 as well as for the SP receptor NK1 (1). Additionally, the possibility has been raised that SP antibodies may cross-react with other tachykinins including NKA and NKB (16). These issues of specificity and cross-reactivity have become troublesome to us as evidence accumulates to suggest that NKA and NKB as well as SP may participate in the spinal transmission of the exercise pressor reflex. This evidence includes the following. First, both NKA and NKB and their binding sites have been isolated in the dorsal horn of the spinal cord (11,21). Second, NKA has been colocalized with SP to the spinal terminals of primary afferents (3). Third, immunoreactive NKA has been detected in the lumbar dorsal horn after isometric contraction of the cat hindlimb (4). Finally, although the mRNA for NKB has not been detected in dorsal root ganglion cells (27), NKB has been localized in spinal interneurons and in ascending pathways (22). SP antagonists specific for the NK-1 receptor have been unavailable until the recent development of the highly specific NK-1 receptor antagonist CP 96,345 (24). This capability of blocking selectively the NK-1 receptor has prompted us to reexamine the role played by SP in
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the spinal transmission of the exercise pressor reflex. Therefore, we tested the hypothesis that, after the intrathecal injection of CP 96,345 onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Additionally, whether the stimulation of NK-2 receptors by NKA and NK-3 receptors by NKB modulates the spinal transmission of the exercise pressor reflex is not known. Therefore, we tested the hypotheses that, after the intrathecal injection of an NK-2 receptor antagonist or an NK-3 receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. METHODS
General. The experiments were performed on adult cats (2.1-6.0 kg). Anesthesia was induced with ketamine hydrochloride (25 mg/kg SC). An external jugular vein was cannulated, and cu-chloralose was injected intravenously (25 mg/kg). Additional cu-chloralose (5 mg/kg iv) was administered whenever the cat exhibited a withdrawal response to a noxious paw pinch or a pressor response to surgical manipulation. The total dose of cychloralose administered ranged from 40 to 75 mg/kg. A common carotid artery was cannulated. The carotid catheter was attached to a Statham transducer (P23 XL) from which arterial blood pressure was measured. Heart rate (HR) was calculated beat to beat (Gould Biotach) from the arterial pressure pulse. A cannula was placed in the trachea and connected to a heated pneumotach (Fleisch no. 00). Inspiratory airflow was integrated (Gould Integrator) breath by breath to give tidal volume from which minute volume was calculated. Throughout the experiment, the cat breathed spontaneously. A laminectomy exposed the lumbar and sacral spinal cord. A small hole was made in the dura at the second lumbar segment. A catheter (PE-50) was passed through the hole and under the arachnoid membrane until the tip of the catheter was positioned at the fifth lumbar segment. The cat was secured in a Kopf spinal unit. A pillow was placed beneath the head and chest regions to elevate the brain and the cervical and thoracic spinal segments above the lumbosacral segment. The rostra1 portion of the cat was elevated to minimize the spread of agents injected through the intrathecal cannula to and above the medulla. The left tibia1 nerve was isolated from the surrounding tissue. A hook electrode was positioned around the tibia1 nerve at the junction of the nerve and the triceps surae muscles. Static contraction of the left triceps surae muscles was evoked by stimulating the tibia1 nerve at 1.5-2.5 times motor threshold, 20-40 Hz, 0.025 ms. This laboratory has shown that group III and IV fibers are not activated electrically when the tibia1 nerve is stimulated at these intensities (12); hence, the reflex cardiovascular and ventilatory responses we measured were attributed to muscle contraction. The left calcaneal tendon was severed at the junction with the calcaneal bone. The severed tendon was connected to a force transducer (Grass FTIO) that measured the developed tension of the contract-
EXERCISE
PRESSOR
REFLEX
1. Effect of neurokin@ receptor antagonists on baseline MAP, HR, and VI TABLE
Drug
n
CP 96,345 (mol wt 485) MAP, mmHg HR, beats/min VI, ml/min [D-Pro2,D-Phe7,D-Trp’]SP (mol wt 1,476.8) MAP, mmHg HR, beats/min VI, mllmin MEN 10.207 (mol wt lJO8.5) MAP, mmHg HR, beats/min VI, ml/min [ D-Pro2,&I’rp6~8~g,N1e10] NKB (mol wt 1,326.5) MAP, mmHg HR, beats/min VI, mllmin
7
Before
After
146+8 216kll 254+44
147s 216kll 264+60
129t6 20927 220t31
136+8 208+8 247+39
142&5 212~17 23Ok24
141+4 212k6 240+47
134t17 2OO-t7 271+14
132215 203+4 203k26
6
6
5
Values are means & SE. n, no. of cats. Neurokinin receptor antagonists were administered by intrathecal inj.ection in lOO-pg doses. MAP, mean arterial pressure; HR, heart rate; VI, minute volume of ventilation; SP, substance P; NKB, neurokinin B.
ing triceps surae muscles. One kilogram of resting tension was applied to the triceps surae muscles. ExperimentaL protocol. A 60-s control period with the triceps surae muscles at rest preceded each 60-s static contraction of the triceps surae muscles. From the 60-s control period, we determined steady-state mean arterial pressure (MAP) and HR, and we calculated minute volume of ventilation (VI) by summing the integrated tidal volumes. From the 60-s period of static contraction, we determined peak MAP and HR, and, again, we calculated VI. We defined a reflex increase in MAP, HR, or VI as the difference between the values obtained during the control period and during exercise. The tension developed by the triceps surae muscles during a 60-s static contraction was calculated as peak tension minus resting tension. Once the reflex cardiovascular and ventilatory responses to static contraction were determined and after MAP and VI had returned to precontraction steady-state levels, an intrathecal injection (0.2 ml) of one of the antagonists was given (Table 1). The following protocols were used for each of the antagonists. NK-1 antagonist: CP 96,345. Ten minutes after the intrathecal injection of CP 96,345, the triceps surae muscles were statically contracted. If the reflex increases in MAP and VI 10 min after CP 96,34 were not attenuated, we tested the accessibility of the antagonist to the parts of the dorsal horn receiving afferent input from the contracting triceps surae muscles. Lidocaine hydrochloride (0.2 ml, 2%) was injected intrathecally. Ten minutes later, the cardiovascular and ventilatory reflex responses to static contraction were reassessed. Failure to attenuate by 50% the reflex cardiovascular and ventilatory adjustments to static contraction after lidocaine indicated that neither the lidocaine nor the antagonist had access to the sites of muscle afferent input to the dorsal horn. The data were excluded from our analysis. Finally, in those cats whose reflex pressor and ventilatory responses to static muscular contraction were attenuated
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NK-1
RECEPTORS
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THE
after the intrathecal injection of the neurokinin receptor antagonist, Evans blue dye (0.2 ml) was injected through the intrathecal cannula onto the lumbosacral spinal cord. Ten minutes later, the rostrocaudal spread of the dye was assessed. In no cat had the dye migrated above L,. NK-1 INK-2 antagonist: [D-Pro2,D-Phe7, D- Trpg]sP. Ten minutes after the intrathecal injection of [D-Pro2,DPhe7,D-Trpg]SP, the triceps surae muscles were statically contracted. The reflex cardiovascular and ventilatory adjustments to static contraction were reassessed 60 min later in those cats in which the reflex pressor response to contraction was attenuated after intrathecal [D-Pro2,D-Phe7,D-Trp’] SP. This later contraction was performed because the attenuating effects of [D-Pr02,DPhe7,D-Trpg]SP on the exercise pressor reflex have been reported to be short lasting (i.e., 25-70 min) after intrathecal injection of this antagonist onto the lumbosacral spinal cord (12). The cats in which intrathecal [D-Pr02,DPhe7,D-Trpg]SP did not attenuate the reflex pressor response to static contraction received an intrathecal injection (0.2 ml) of 2% lidocaine hydrochloride (see above). Finally, Evans blue dye (0.2 ml) was injected (see above). NK-2 and NK-3 antagonists. Ten minutes after the intrathecal injection of either MEN 10.207 or Nle”, the triceps surae muscles were statically contracted. After intrathecal MEN 10.207, some of these cats received an intrathecal injection of [D-Pro2,D-Phe7,D-Trp’]SP while the others received an intrathecal injection of 2% lidoCaine hydrochloride (0.2 ml). Ten minutes later, the reflex increases in MAP, HR, and VI were reassessed. Evans blue dye (0.2 ml) was injected intrathecally (see above). Drugs. The volume of each intrathecal injection was 0.2 ml. The intrathecal injection of 0.2 ml saline has been shown to have no effect on the exercise pressor reflex (12). Each intrathecal injection was flushed into the cerebrospinal fluid with normal saline in a volume equal to the dead space of the intrathecal cannula (0.06-0.09 ml). The antagonists were dissolved in normal saline. The neurokinin receptor antagonists and doses used were as follows: NK-1 receptor antagonist: 10,50, and 100 pg CP 96,345 (24); NK-l/NK-2 receptor antagonist: 100 pg [DPro2,D-Phe7,D-Trpg]SP (16); NK-2 receptor antagonist: 100 and 500 pg [Tyr5,D-Trp6*8~gArg10]NKA-(4 - 10) (MEN 10.207) (15); NK-3 receptor antagonist: 100 pg [D-Pro2,D-Trp6~8Nle10]NKB (Nle”) (26). Data analysis. All data are reported as means t SE. A repeated-measures analysis of variance was used to determine overall significance. When appropriate, a Dunnett’s post hoc test was calculated (20). The criterion for statistical significance was P < 0.05. RESULTS
General. None of the neurokinin receptor antagonists injected intrathecally (100 ,ug; 0.2 ml) onto the lumbosacral spinal cord altered the levels of MAP, HR, and VI measured while the triceps surae muscles were at rest (Table 1). NK-1 receptor antagonists. Static contractions of the triceps surae muscles of seven cats sienificantlv in-
EXERCISE
PRESSOR
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1391
creased (P < 0.05) MAP (27 t 7 mmHg) and VI (181 t 52 ml/min), but not HR (11 t 3 beats/min), over control levels. These reflex pressor and ventilatory responses to static muscular contraction were significantly attenuated (P < 0.05) by 100 pugCP 96,345 (Figs. 1 and 2). The tensions developed by the contracting triceps surae muscles averaged 4.2 t 0.6 kg before CP 96,345 and 4.1 t 0.6 kg after CP 96,345. We examined the effects of 10 pg and a total of 50 pg CP 96,345 on the pressor and ventilatory responses to static muscular contraction. In six cats, before CP 96,345 was injected intrathecally the cardiovascular and ventilatory responses to static contraction were increased over steady-state levels by 22 t 2 mmHg, 7 + 3 beats/ min, and 159 t 63 ml/min (MAP, HR, and VI, respectively). Tensions developed by the contracting triceps surae muscles averaged 4.5 t 0.6 kg. Next, 10 pg CP 96,345 were injected intrathecally. Ten minutes later, static muscular contraction evoked reflex increases in MAP (24 t 3 mmHg), HR (8 t 3 beats/min), and VI (122 +- 33 ml/min) that were not different (P > 0.05) from the corresponding preantagonist increases. Tensions developed by the contracting triceps surae muscles averaged 4.3 + 0.7 kg. The cardiovascular and ventilatory reflex respinses to static contraction were then measured 10 min after a total dose of 50 pg CP 96,345 was injected intrathecally. Compared with preantagonist increases, contraction-induced reflex increases in MAP (17 t 3 mmHg), HR (4 t 1 beats/min), and VI (121 t 40 ml/min) were unaffected (P > 0.05) by the total dose of 50 pg CP 96,345. Tensions developed by the contracting triceps surae muscles averaged 4.2 t 0.6 kg. Finally, lidocaine was injected intrathecally. Ten minutes later, the contraction-induced reflex pressor and ventilatory responses were significantly (P < 0.05) reduced, with MAP increasing by only 8 t 1 mmHg and VI by only 68 t 59 ml/min. The increase in HR (6 t 2 beats/min) was not attenuated (P > 0.05) after lidocaine. The tensions developed by the contracting triceps surae muscles averaged 4.1 t 0.6 kg. In six cats the reflex pressor response to static muscular contraction was significantly (P < 0.05) attenuated 10 min after 100 pg [D-Pro2,D-Phe7,D-Trp’]SP was injected intrathecally (Fig. 3). The chronotropic and ventilatory responses, although less than preinjection levels, were not significantly (P > 0.05) attenuated (Fig. 3). Sixty minutes after 100 pg [D-Pro2,D-Phe7,D-Trp’]SP had been injected intrathecally, the pressor response to static contraction was restored (Fig. 3). The developed tensions recorded for the three contractions were 5.1 t 0.9, 5.2 t 0.9, and 5.2 t 0.8 kg, respectively. NK-2 antagonist. In six cats the reflex cardiovascular and ventilatory responses to static muscular contraction were measured before the intrathecal injection of the NK-2 receptor antagonist MEN 10.207,lO min after 100 pg MEN 10.207, and 10 min after 100 pg [D-Pr02,1?Phe7,D-Trpg]SP. Reflex increases in MAP, HR, and VI before MEN 10.207 were 29 t 4 mmHg, 11 t 1 beats/min, 114 t 74 ml/ min, respectively. After 100 pg MEN 10.207, these increases were 30 t 6 mmHg, 10 t 1 beats/min, and 137 t 89 ml/ min, respectively. Finally, 10 min after 100 pg [D-Pro2,D-Phe7,D-Trp’] SP, the reflex responses to contraction were 20 t 6 mmHg. 7 t 2 beats/min, and 74
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9~~[~
Lfl
RECEPTORS
A,
AND THE EXERCISE
(J
n
PRESSOR
fl
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m
FIG. 1. Effects of intrathecal CP 96,345 (100 pg) on contraction-induced reflex increases in arterial blood pressure (BP), heart rate (HR), and tidal volume (VT) in 1 cat. A: reflex pressor, chronotropic, and ventilatory responses to static contraction before CP 96,345. B: reflex responses to static contraction 10 min after CP 96,345. Pressor and ventilatory responses to static contraction are attenuated after CP 96,345. Tensions developed by triceps surae muscles for each contraction are similar.
+ 72 ml/min, respectively. The reflex increase in MAP after [D-Pro2,D-Phe7,D-Trp’]SP was significantly (P < 0.05) attenuated compared with the reflex increase in MAP before MEN 10.207. The chronotropic and ventilatory responses to contraction after [D-Pro2,D-Phe7,DTrpg]SP were not different (P > 0.05) from those before MEN 10.207. Likewise, the pressor, chronotropic, and ventilatory reflex responses after MEN 10.207 were not
different (P > 0.05) from those before MEN 10.207. The developed tensions for the three contractions were 4.5 f 0.8, 4.5 + 0.8, and 4.4 ~lt0.8 kg, respectively. In three other cats, we increased the dose of intrathecal MEN 10.207 to 500 pg. Contraction-induced reflex increases in MAP and HR before and after 500 hg MEN 10.207 were identical, 41 ?z 15 mmHg and 2 t 2 beats/min and 41 + 15 mmHg and 2 + 2 beats/min, respectively. 240
15r
2
a e
2
180
-
160
z
.;
140
%
z
120
-
100
w
::
'0
5
80
5
60
3
40 20 0
0 II=1
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n=7
2. Summary data for cardiovascular and ventilatory responses of 7 cats to static contraction of triceps surae muscles before (filled columns) and 16 min after (hatched columns) CP 96,345 (means + SE). Reflex changes in MAP and minute volume of ventilation (VI) are significantly attenuated after CP 96,345. Reflex change in HR is not significantly attenuated. * Significantly different (P < 0.05). FIG.
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NK-1 RECEPTORS
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PRESSOR REFLEX 250
T
T
=
225
‘ii
200
: e
175
-5 150 z 125 ” too z 75 4 50 E 25 0 n=6
n=6
a-6
FIG. 3. Effects of intrathecal injection of 100 rg [D-Pro2,D-Phe7,D-Trp’]SP (D-Pro) on reflex cardiovascular and ventilatory responses to static contraction before (closed columns), 10 min after (hatched columns), and >30 min after (open columns) ~-Pro (means 2 SE). Reflex change in MAP is significantly attenuated after ~-Pro. More than 30 min later, reflex change in MAP has returned to level achieved before D-Pro. Reflex changes in HR and %‘I are not significantly attenuated after D-Pro. * Significantly different (P < 0.05).
Before MEN 10.207 the ventilatory response to contraction was 279 + 62 ml/min, whereas after 500 pg MEN 10.207 this response was 222 2 60 ml/min (P > 0.05). The pressor and ventilatory responses to static contraction were significantly attenuated (P < 0.05) after intrathecal lidocaine (17 f 3 mmHg and 53 f 3 ml/min, respectively). Before MEN 10.207, after MEN 10.207, and after lidocaine, the developed tensions of the triceps surae muscles averaged 6.1 f 0.6, 5.6 + 1.1, and 5.7 + 0.8 kg, respectively.
NK-3 antagonist. In five cats the reflex pressor, chronotropic, and ventilatory responses to static contraction of the triceps surae muscles were measured before and 10 min after the intrathecal injection of the NK-3 antagonist Nle”. Before Nle”, static contraction evoked increases in MAP, HR, and VI of 19 f 1 mmHg, 12 + 2 beats/min, and 106 + 42 ml/min, respectively. After Nlel’, reflex increases in MAP, HR, and VI averaged 24 + 4 mmHg, 14 + 3 beats/min, and 203 + 37 ml/min, respectively. Contraction-induced reflex increases in MAP, HR, and VI before Nlel’ and after Nle” were not
significantly different (P > 0.05). The developed tensions of the triceps surae muscles averaged 5.6 + 1 and 5.6 -t 1 kg. DISCUSSION
We have shown that the injection of the selective NK-1 receptor antagonist CP 96,345 into the cerebrospinal fluid bathing the lumbosacral
spinal cord attenuated
the
reflex pressor and ventilatory responses to static contraction of the triceps surae muscles. In addition, we confirmed a previous finding that the reflex pressor response to static muscular contraction is attenuated by [DPro’,D-Phe7,D-Trpg]SP, an NK-l/NK-2 receptor antagonist (12). We were unable to provide evidence that NK-2 or NK-3 receptors are involved in the spinal transmission of the exercise pressor reflex. We do not believe that the attenuating effects of CP 96,345 on the exercise pressor reflex can be explained by a neurotoxic effect of this antagonist. McLean et al. (17) showed that the application of CP 96,345 to the medium perfusing locus ceruleus neurons did not alter their baseline firing rates. In addition, we do not believe that our
results are a consequence of the effect of CP 96,345 on sympathetic outflow. Using Evans blue dye as an index of the extent of spread of an injectate,
we showed that the
thoracic spinal segments, in which sympathetic outflow to the heart and lungs originates, were not stained blue after the intrathecal
injection
of Evans blue dye onto
the lumbosacral spinal cord. Moreover, we previously showed that sympathetic outflow, as assessedby the cardiovascular
and ventilatory
responses either to electrical
stimulation of sciatic nerve afferents synapsing in the upper lumbar cord (7-9) or to electrical stimulation of the posterior hypothalamus (13) was not affected by substances injected onto the lower lumbar and sacral spinal cord. A role for NKA in the spinal transmission of the exercise pressor reflex has yet to be defined. Recently, NKA
was detected in the dorsal horn of the spinal cord after repeated isometric contractions of the feline hindlimb muscles (4). This finding suggested to us that NKA may play a neurotransmitter/modulatory
role in the exercise
pressor reflex. Our attempts to demonstrate such a role were unsuccessful. The reflex cardiovascular and ventilatory
responses to a static muscular
contraction
were
unaffected after the intrathecal injection of the NK-2 receptor antagonist MEN 10.207. This lack of effect by MEN 10.207 on the exercise pressor reflex probably was not a consequence of the inaccessibility of MEN 10.207 to the appropriate spinal laminae of the dorsal horn, because
[D-Pro2,D-Phe7,D-Trp’]SP
or lidocaine,
which
were injected intrathecally through the same cannula and in the same volume as MEN 10.207, attenuated the reflex responses to static contraction.
Nevertheless,
we
cannot exclude the possibility that MEN 10.207 was ineffective because of incomplete blockade of NK-2 receptors in the dorsal horn. Although
we injected intrathe-
tally lOO- and 500-pg doses of MEN 10.207, a sufficient number of NK-2 receptors may have remained available for NKA activation. We speculate that NKA functioning as an endogenous ligand for NK-2 receptors in the dorsal horn does not participate in the spinal transmission of the exercise pressor reflex. In our experiments, only NK-1 receptor blockade, with either the specific NK-1 receptor antago-
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nist or the combined NK-l/NK-2 receptor antagonist, attenuated the pressor and ventilatory responses to contraction. If NKA is a spinal neuromodulator of the exercise pressor reflex, NKA probably exerts its effects through the SP receptor NK-1. Endogenous NKA has been reported to be a ligand for the NK-1 and NK-2 receptors (23). The functional significance for the existence of NK-3 receptors in the dorsal horn is yet to be elucidated. Conflicting evidence has been presented concerning the involvement of NK-3 receptors in the spinal processing of somatosensory information. For example, Laneuville et al. (14) found that intrathecal NKB increased reaction time to a radiant heat stimulus. Conversely, FleetwoodWalker et al. (5) found that the responses of spinal cervical tract neurons to noxious stimuli were unaffected after the microiontophoretic application of either a NK-3 receptor agonist, Senkitide, or antagonist, Nle”. We, too, were unable to identify a physiological role for spinal NK-3 receptors, hence, our finding that the cardiovascular and ventilatory responses to static muscular contraction were unaffected after the intrathecal injection of the NK-3 receptor antagonist Nlel’. SP has been identified as a putative neurotransmitter or neuromodulator of contraction-activated afferent input to the dorsal horn of the spinal cord. Two pieces of evidence point to neuromodulation rather than to neurotransmission as the role played by SP. First, SP has been shown to induce a slow depolarization of dorsal horn neurons (25). Slow depolarization is characteristic of a modulator of afferent input to the dorsal horn, whereas rapid synaptic action is characteristic of a transmitter (2). Second, exogenous SP microinjected into the feline L, dorsal horn with the hindlimb muscles at rest has been shown to have no effect on blood pressure or HR (28). If SP had acted as a neurotransmitter, the cardiovascular changes that occur with static exercise should have been mimicked by the application of SP into the dorsal horn. We have tested three hypotheses in an attempt to identify a role for each of three related tachykinins, SP, NKA, and NKB, in the spinal transmission of the exercise pressor reflex. Using NK-2 (NKA) and NK-3 (NKB) receptor antagonists, we were unable to show any change in the cardiovascular or ventilatory responses to static muscular contraction. Using an NK-1 (SP) receptor antagonist, we were able to attenuate the pressor and ventilatory responses to static muscular contraction. Our findings are good evidence linking SP and NK-1 receptors with the exercise pressor reflex. We speculate that the role played by SP is one of spinal neuromodulation of this reflex. We thank Charles Stuart for technical assistance. We also thank Pfizer, Inc., for the generous gift of CP 96,345. This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-30710. J. M. Hill was supported by American Heart Association, California Affiliate, Fellowship 90-17. J. G. Pickar was supported by NHLBI National Research Service Award HL-08144. Address for reprint requests: J. M. Hill, Div. of Cardiovascular Medicine, TB172, University of California, Davis, CA 95616. Received
16 December
1991; accepted
in final
form
28 April
EXERCISE
3.
4.
5.
6. 7.
8.
9.
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16. 17.
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22.
1992.
REFERENCES 1. BUCK, S. H., AND S. A. SHATZER. Agonists and antagonists binding to tachykinin NK-2 receptors. Life Sci. 42: 2701-2708, 1988. 2. CHRISTENSEN, B. N., AND E. R. PERL. Spinal neurons specifically
23. 24.
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excited by noxious or thermal stimuli: marginal zone of the dorsal horn. J. Neurophysiol. 33: 293-307, 1977. DALSGAARD, C.-J., A. HAEGERSTRAND, E. THEODORSSON-NORHEIM, E. BRODIN, AND T. HOKFELT. Neurokinin A-like immunoreactivity in rat primary sensory neurons: coexistence with substance P. Histochemistry 83: 37-40, 1985. DUGGAN, A. W., P. J. HOPE, C. W. LANG, AND C. A. WILLIAMS. Sustained isometric contraction of skeletal muscle results in release of immunoreactive neurokinins in the spinal cord of the anaesthetized cat. Neurosci. Lett. 122: 191-194, 1991. FLEETWOOD-WALKER, S. M., R. MITCHELL, P. J. HOPE, N. ELYASSIR, V. MOLONY, AND C. M. BLADON. The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat. Bruin Res. 519: 169-182, 1990. HENRY, J. L. Effects of substance P on functionally identified units in cat spinal cord. Bruin Res. 114: 439-451, 1976. HILL, J. M., AND M. P. KAUFMAN. Attenuation of reflex pressor and ventilatory responses to static muscular contraction by intrathecal opioids. J. Appl. Physiol. 68: 2466-2472, 1990. HILL, J. M., AND M. P. KAUFMAN. Attenuating effects of intrathecal clonidine on the exercise pressor reflex. J. Appl. Physiol. 70: 516-522, 1991. HILL, J. M., AND M. P. KAUFMAN. Intrathecal serotonin attenuates the pressor response to static contraction. Brain Res. 550: 157-160, 1991. HOKFELT, T., R. ELDE, 0. JOHANSSON, R. LUFT, G. NILSSON, AND A. ARIMURA. Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat. Neuroscience 1: 131-136, 1976. KANAZAWA, I., T. OGAWA, S. KIMURA, AND E. MUNEKATA. Regional distribution of substance P, neurokinin A and neurokinin B in rat central nervous system. Neurosci. Res. 2: 111-120, 1984. KAUFMAN, M. P., G. P. KOZLOWSKI, AND K. J. RYBICKI. Attenuation of the reflex pressor response to muscular contraction by a substance P antagonist. Brain Res. 333: 182-184, 1985. KAUFMAN, M. P., K. J. RYBICKI, G. P. KOZLOWSKI, AND G. I. IWAMOTO. Immunoneutralization of substance P attenuates the reflex pressor response to muscular contraction. Bruin Res. 377: 199-203, 1986. LANEUVILLE, O., J. DORAIS, AND R. COUTURE. Characterization of the effects produced by neurokinins and three agonists selective for neurokinin receptor subtypes in a spinal nociceptive reflex of the rat. Life Sci. 42: 1295-1305, 1988. MAGGI, C. A., R. PATACCHINI, S. GIULIANI, P. ROVERO, S. DION, D. REGOLI, A. GIACHETTI, AND A. MELI. Competitive antagonists discriminate between NK, tachykinin subtypes. Br. J. Pharmacol. 100: 588-592, 1990. MAGGIO, J. E. Tachykinins. Annu. Rev. Neurosci. 11: 13-28, 1988. MCLEAN, S., A. H. GANONG, T. F. SEEGER, D. K. BRYCE, K. G. PRATT, L. S. REYNOLDS, C. J. SIOK, J. A. LOWE III, AND J. HEYM. Activity and distribution of binding sites in brain of a nonpeptide substance P (NKl) receptor antagonist. Science Wash. DC 251: 437-439, 1991. MCCLOSKEY, D. I., AND J. H. MITCHELL. Reflex cardiovascular and respiratory responses originating in exercising muscle. J. Physiol. Lond. 224: 172-186, 1972. MITCHELL, J. H., M. P. KAUFMAN, AND G. W. IWAMOTO. The exercise pressor reflex: its cardiovascular effects, afferent mechanisms, and central pathways. Annu. Rev. Physiol. 45: 229-242, 1983. MYERS, J. L. Fundamentals of Experimental Design. Boston, MA: Allyn and Bacon, 1966. O’DONOHUE, T. L., C. J. HELKE, E. BURCHER, C. W. SHULTS, AND S. H. BUCK. Autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system. In: Substance P and Neurokinins, edited by J. L. Henry, R. Couture, A. C. Cuello, G. Pelletier, R. Quirion, and D. Regoli. New York: Springer, 1987, p. 93-95. OGAWA, T., I. KANAZAWA, AND S. KIMURA. Regional distribution of substance P, neurokinin A, neurokinin B in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection. Bruin Res. 359: 152-157, 1985. QUIRION, R., AND T. V. DAM. Multiple neurokinin receptors: recent developments. Regul. Pept. 22: 18, 1988. SNIDER, R. M., J. W. CONSTANTINE, J. A. LOWE III, K. P. LONGO, W. S. LEBEL, H. A. WOODY, S. E. DROZDA, M. C. DESAI, F. J. VINICH, R. W. SPENCER, AND H. HESS. A potent nonpeptide antag-
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NK-1
RECEPTORS
AND
THE
onist of the substance P (NKl) receptor. Science Wash. DC 251: 435-437,199l. 25. URBAN, L., AND M. RANDIC. Slow excitatory transmission in rat dorsal horn: possible mediation by peptides. Brain Res. 290: 336341,1984. 26. VAUGHT, J.L.,R.SCOTT,D. WRIGHT,AND H. JACOBY. [D-Pro2,DTrp6~8,Nle’o] -neurokinin B: pharmacological profile of a novel neurokinin antagonist. In: Substance P and Neurokinins, edited by J. L. Henry, R. Couture, A. C. Cuello, G. Pelletier, R. Quirion, and D. Regoli. New York: Springer, 1987, p. 120-124.
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27. WARDEN, M. K., AND W. S. YOUNG. Distribution of cells containing mRNA’s encoding substance P and neurokinin B in the rat central nervous system. J. Camp. Neurol. 272: 90-113, 1988. 28. WILSON, L.B.,P.T. WALL, K. MATSUKAWA,AND J.H. MITHCELL. Effect of spinal microinjections of an antagonist to substance P or somatostatin on the exercise pressor reflex. Circ. Res. 70: 213-222, 1992. 29. YAKSH, T.L.,T.M. JESSELL, R. GAMSE, A.W. MUDGE,ANDS. E. LEEMAN. Intrathecal morphine inhibits substance P release from mammalian spinal cord, in vivo. Nature Lond. 286: 155-157, 1980.
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substance
P; CP 96,345; control
of
THEEXERCISEPRESSORREFLEX referstothe reflexcardiovascular and ventilatory increases evoked by static muscular contraction (19). The afferent arm of this reflex arc is comprised of group III and IV fibers, which are innervated by sensory endings in the contracting muscle and which synapse in the dorsal horn of the spinal cord (18). The efferent arm of this reflex arc is comprised of the sympathetic outflow to the heart and blood vessels, the parasympathetic nerves to the heart, and the motor nerves to the respiratory muscles. 0161-7567/92 $2.00 Copyright
The chemical messengers released onto the secondorder dorsal horn neurons from the spinal terminals of contraction-activated group III and IV fibers have not been determined. One important candidate is the tachykinin substance P (SP), which has a role as a spinal neurotransmitter/modulator in the exercise pressor reflex that has been suggested by the findings of several studies. For example, SP has been found in the spinal terminals of fine-caliber axons running in the L,--S, dorsal roots (10). SP has been shown to be released in the spinal cord when A& and C-fibers in the sciatic nerve have been electrically stimulated (29). Iontophoresis of SP has been shown to stimulate dorsal horn cells receiving somatic afferent input (6). Finally, SP receptors [neurokinin-1 (NK-l)] have been identified immunohistochemically in the dorsal horn (21). Three previous studies have demonstrated an attenuated pressor response to static muscular contraction after the microinjection of a SP antagonist into the L, dorsal horn (28) or after the intrathecal injection of a SP antagonist (12) or antibody (13) onto the feline lumbosacral spinal cord. Since these findings, the antagonist used in both the microinjection and intrathecal studies has been shown to exhibit an affinity for the neurokinin A (NKA) receptor NK-2 as well as for the SP receptor NK1 (1). Additionally, the possibility has been raised that SP antibodies may cross-react with other tachykinins including NKA and NKB (16). These issues of specificity and cross-reactivity have become troublesome to us as evidence accumulates to suggest that NKA and NKB as well as SP may participate in the spinal transmission of the exercise pressor reflex. This evidence includes the following. First, both NKA and NKB and their binding sites have been isolated in the dorsal horn of the spinal cord (11,21). Second, NKA has been colocalized with SP to the spinal terminals of primary afferents (3). Third, immunoreactive NKA has been detected in the lumbar dorsal horn after isometric contraction of the cat hindlimb (4). Finally, although the mRNA for NKB has not been detected in dorsal root ganglion cells (27), NKB has been localized in spinal interneurons and in ascending pathways (22). SP antagonists specific for the NK-1 receptor have been unavailable until the recent development of the highly specific NK-1 receptor antagonist CP 96,345 (24). This capability of blocking selectively the NK-1 receptor has prompted us to reexamine the role played by SP in
0 1992 the American
Physiological
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the spinal transmission of the exercise pressor reflex. Therefore, we tested the hypothesis that, after the intrathecal injection of CP 96,345 onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Additionally, whether the stimulation of NK-2 receptors by NKA and NK-3 receptors by NKB modulates the spinal transmission of the exercise pressor reflex is not known. Therefore, we tested the hypotheses that, after the intrathecal injection of an NK-2 receptor antagonist or an NK-3 receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. METHODS
General. The experiments were performed on adult cats (2.1-6.0 kg). Anesthesia was induced with ketamine hydrochloride (25 mg/kg SC). An external jugular vein was cannulated, and cu-chloralose was injected intravenously (25 mg/kg). Additional cu-chloralose (5 mg/kg iv) was administered whenever the cat exhibited a withdrawal response to a noxious paw pinch or a pressor response to surgical manipulation. The total dose of cychloralose administered ranged from 40 to 75 mg/kg. A common carotid artery was cannulated. The carotid catheter was attached to a Statham transducer (P23 XL) from which arterial blood pressure was measured. Heart rate (HR) was calculated beat to beat (Gould Biotach) from the arterial pressure pulse. A cannula was placed in the trachea and connected to a heated pneumotach (Fleisch no. 00). Inspiratory airflow was integrated (Gould Integrator) breath by breath to give tidal volume from which minute volume was calculated. Throughout the experiment, the cat breathed spontaneously. A laminectomy exposed the lumbar and sacral spinal cord. A small hole was made in the dura at the second lumbar segment. A catheter (PE-50) was passed through the hole and under the arachnoid membrane until the tip of the catheter was positioned at the fifth lumbar segment. The cat was secured in a Kopf spinal unit. A pillow was placed beneath the head and chest regions to elevate the brain and the cervical and thoracic spinal segments above the lumbosacral segment. The rostra1 portion of the cat was elevated to minimize the spread of agents injected through the intrathecal cannula to and above the medulla. The left tibia1 nerve was isolated from the surrounding tissue. A hook electrode was positioned around the tibia1 nerve at the junction of the nerve and the triceps surae muscles. Static contraction of the left triceps surae muscles was evoked by stimulating the tibia1 nerve at 1.5-2.5 times motor threshold, 20-40 Hz, 0.025 ms. This laboratory has shown that group III and IV fibers are not activated electrically when the tibia1 nerve is stimulated at these intensities (12); hence, the reflex cardiovascular and ventilatory responses we measured were attributed to muscle contraction. The left calcaneal tendon was severed at the junction with the calcaneal bone. The severed tendon was connected to a force transducer (Grass FTIO) that measured the developed tension of the contract-
EXERCISE
PRESSOR
REFLEX
1. Effect of neurokin@ receptor antagonists on baseline MAP, HR, and VI TABLE
Drug
n
CP 96,345 (mol wt 485) MAP, mmHg HR, beats/min VI, ml/min [D-Pro2,D-Phe7,D-Trp’]SP (mol wt 1,476.8) MAP, mmHg HR, beats/min VI, mllmin MEN 10.207 (mol wt lJO8.5) MAP, mmHg HR, beats/min VI, ml/min [ D-Pro2,&I’rp6~8~g,N1e10] NKB (mol wt 1,326.5) MAP, mmHg HR, beats/min VI, mllmin
7
Before
After
146+8 216kll 254+44
147s 216kll 264+60
129t6 20927 220t31
136+8 208+8 247+39
142&5 212~17 23Ok24
141+4 212k6 240+47
134t17 2OO-t7 271+14
132215 203+4 203k26
6
6
5
Values are means & SE. n, no. of cats. Neurokinin receptor antagonists were administered by intrathecal inj.ection in lOO-pg doses. MAP, mean arterial pressure; HR, heart rate; VI, minute volume of ventilation; SP, substance P; NKB, neurokinin B.
ing triceps surae muscles. One kilogram of resting tension was applied to the triceps surae muscles. ExperimentaL protocol. A 60-s control period with the triceps surae muscles at rest preceded each 60-s static contraction of the triceps surae muscles. From the 60-s control period, we determined steady-state mean arterial pressure (MAP) and HR, and we calculated minute volume of ventilation (VI) by summing the integrated tidal volumes. From the 60-s period of static contraction, we determined peak MAP and HR, and, again, we calculated VI. We defined a reflex increase in MAP, HR, or VI as the difference between the values obtained during the control period and during exercise. The tension developed by the triceps surae muscles during a 60-s static contraction was calculated as peak tension minus resting tension. Once the reflex cardiovascular and ventilatory responses to static contraction were determined and after MAP and VI had returned to precontraction steady-state levels, an intrathecal injection (0.2 ml) of one of the antagonists was given (Table 1). The following protocols were used for each of the antagonists. NK-1 antagonist: CP 96,345. Ten minutes after the intrathecal injection of CP 96,345, the triceps surae muscles were statically contracted. If the reflex increases in MAP and VI 10 min after CP 96,34 were not attenuated, we tested the accessibility of the antagonist to the parts of the dorsal horn receiving afferent input from the contracting triceps surae muscles. Lidocaine hydrochloride (0.2 ml, 2%) was injected intrathecally. Ten minutes later, the cardiovascular and ventilatory reflex responses to static contraction were reassessed. Failure to attenuate by 50% the reflex cardiovascular and ventilatory adjustments to static contraction after lidocaine indicated that neither the lidocaine nor the antagonist had access to the sites of muscle afferent input to the dorsal horn. The data were excluded from our analysis. Finally, in those cats whose reflex pressor and ventilatory responses to static muscular contraction were attenuated
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NK-1
RECEPTORS
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THE
after the intrathecal injection of the neurokinin receptor antagonist, Evans blue dye (0.2 ml) was injected through the intrathecal cannula onto the lumbosacral spinal cord. Ten minutes later, the rostrocaudal spread of the dye was assessed. In no cat had the dye migrated above L,. NK-1 INK-2 antagonist: [D-Pro2,D-Phe7, D- Trpg]sP. Ten minutes after the intrathecal injection of [D-Pro2,DPhe7,D-Trpg]SP, the triceps surae muscles were statically contracted. The reflex cardiovascular and ventilatory adjustments to static contraction were reassessed 60 min later in those cats in which the reflex pressor response to contraction was attenuated after intrathecal [D-Pro2,D-Phe7,D-Trp’] SP. This later contraction was performed because the attenuating effects of [D-Pr02,DPhe7,D-Trpg]SP on the exercise pressor reflex have been reported to be short lasting (i.e., 25-70 min) after intrathecal injection of this antagonist onto the lumbosacral spinal cord (12). The cats in which intrathecal [D-Pr02,DPhe7,D-Trpg]SP did not attenuate the reflex pressor response to static contraction received an intrathecal injection (0.2 ml) of 2% lidocaine hydrochloride (see above). Finally, Evans blue dye (0.2 ml) was injected (see above). NK-2 and NK-3 antagonists. Ten minutes after the intrathecal injection of either MEN 10.207 or Nle”, the triceps surae muscles were statically contracted. After intrathecal MEN 10.207, some of these cats received an intrathecal injection of [D-Pro2,D-Phe7,D-Trp’]SP while the others received an intrathecal injection of 2% lidoCaine hydrochloride (0.2 ml). Ten minutes later, the reflex increases in MAP, HR, and VI were reassessed. Evans blue dye (0.2 ml) was injected intrathecally (see above). Drugs. The volume of each intrathecal injection was 0.2 ml. The intrathecal injection of 0.2 ml saline has been shown to have no effect on the exercise pressor reflex (12). Each intrathecal injection was flushed into the cerebrospinal fluid with normal saline in a volume equal to the dead space of the intrathecal cannula (0.06-0.09 ml). The antagonists were dissolved in normal saline. The neurokinin receptor antagonists and doses used were as follows: NK-1 receptor antagonist: 10,50, and 100 pg CP 96,345 (24); NK-l/NK-2 receptor antagonist: 100 pg [DPro2,D-Phe7,D-Trpg]SP (16); NK-2 receptor antagonist: 100 and 500 pg [Tyr5,D-Trp6*8~gArg10]NKA-(4 - 10) (MEN 10.207) (15); NK-3 receptor antagonist: 100 pg [D-Pro2,D-Trp6~8Nle10]NKB (Nle”) (26). Data analysis. All data are reported as means t SE. A repeated-measures analysis of variance was used to determine overall significance. When appropriate, a Dunnett’s post hoc test was calculated (20). The criterion for statistical significance was P < 0.05. RESULTS
General. None of the neurokinin receptor antagonists injected intrathecally (100 ,ug; 0.2 ml) onto the lumbosacral spinal cord altered the levels of MAP, HR, and VI measured while the triceps surae muscles were at rest (Table 1). NK-1 receptor antagonists. Static contractions of the triceps surae muscles of seven cats sienificantlv in-
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PRESSOR
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1391
creased (P < 0.05) MAP (27 t 7 mmHg) and VI (181 t 52 ml/min), but not HR (11 t 3 beats/min), over control levels. These reflex pressor and ventilatory responses to static muscular contraction were significantly attenuated (P < 0.05) by 100 pugCP 96,345 (Figs. 1 and 2). The tensions developed by the contracting triceps surae muscles averaged 4.2 t 0.6 kg before CP 96,345 and 4.1 t 0.6 kg after CP 96,345. We examined the effects of 10 pg and a total of 50 pg CP 96,345 on the pressor and ventilatory responses to static muscular contraction. In six cats, before CP 96,345 was injected intrathecally the cardiovascular and ventilatory responses to static contraction were increased over steady-state levels by 22 t 2 mmHg, 7 + 3 beats/ min, and 159 t 63 ml/min (MAP, HR, and VI, respectively). Tensions developed by the contracting triceps surae muscles averaged 4.5 t 0.6 kg. Next, 10 pg CP 96,345 were injected intrathecally. Ten minutes later, static muscular contraction evoked reflex increases in MAP (24 t 3 mmHg), HR (8 t 3 beats/min), and VI (122 +- 33 ml/min) that were not different (P > 0.05) from the corresponding preantagonist increases. Tensions developed by the contracting triceps surae muscles averaged 4.3 + 0.7 kg. The cardiovascular and ventilatory reflex respinses to static contraction were then measured 10 min after a total dose of 50 pg CP 96,345 was injected intrathecally. Compared with preantagonist increases, contraction-induced reflex increases in MAP (17 t 3 mmHg), HR (4 t 1 beats/min), and VI (121 t 40 ml/min) were unaffected (P > 0.05) by the total dose of 50 pg CP 96,345. Tensions developed by the contracting triceps surae muscles averaged 4.2 t 0.6 kg. Finally, lidocaine was injected intrathecally. Ten minutes later, the contraction-induced reflex pressor and ventilatory responses were significantly (P < 0.05) reduced, with MAP increasing by only 8 t 1 mmHg and VI by only 68 t 59 ml/min. The increase in HR (6 t 2 beats/min) was not attenuated (P > 0.05) after lidocaine. The tensions developed by the contracting triceps surae muscles averaged 4.1 t 0.6 kg. In six cats the reflex pressor response to static muscular contraction was significantly (P < 0.05) attenuated 10 min after 100 pg [D-Pro2,D-Phe7,D-Trp’]SP was injected intrathecally (Fig. 3). The chronotropic and ventilatory responses, although less than preinjection levels, were not significantly (P > 0.05) attenuated (Fig. 3). Sixty minutes after 100 pg [D-Pro2,D-Phe7,D-Trp’]SP had been injected intrathecally, the pressor response to static contraction was restored (Fig. 3). The developed tensions recorded for the three contractions were 5.1 t 0.9, 5.2 t 0.9, and 5.2 t 0.8 kg, respectively. NK-2 antagonist. In six cats the reflex cardiovascular and ventilatory responses to static muscular contraction were measured before the intrathecal injection of the NK-2 receptor antagonist MEN 10.207,lO min after 100 pg MEN 10.207, and 10 min after 100 pg [D-Pr02,1?Phe7,D-Trpg]SP. Reflex increases in MAP, HR, and VI before MEN 10.207 were 29 t 4 mmHg, 11 t 1 beats/min, 114 t 74 ml/ min, respectively. After 100 pg MEN 10.207, these increases were 30 t 6 mmHg, 10 t 1 beats/min, and 137 t 89 ml/ min, respectively. Finally, 10 min after 100 pg [D-Pro2,D-Phe7,D-Trp’] SP, the reflex responses to contraction were 20 t 6 mmHg. 7 t 2 beats/min, and 74
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Lfl
RECEPTORS
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AND THE EXERCISE
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FIG. 1. Effects of intrathecal CP 96,345 (100 pg) on contraction-induced reflex increases in arterial blood pressure (BP), heart rate (HR), and tidal volume (VT) in 1 cat. A: reflex pressor, chronotropic, and ventilatory responses to static contraction before CP 96,345. B: reflex responses to static contraction 10 min after CP 96,345. Pressor and ventilatory responses to static contraction are attenuated after CP 96,345. Tensions developed by triceps surae muscles for each contraction are similar.
+ 72 ml/min, respectively. The reflex increase in MAP after [D-Pro2,D-Phe7,D-Trp’]SP was significantly (P < 0.05) attenuated compared with the reflex increase in MAP before MEN 10.207. The chronotropic and ventilatory responses to contraction after [D-Pro2,D-Phe7,DTrpg]SP were not different (P > 0.05) from those before MEN 10.207. Likewise, the pressor, chronotropic, and ventilatory reflex responses after MEN 10.207 were not
different (P > 0.05) from those before MEN 10.207. The developed tensions for the three contractions were 4.5 f 0.8, 4.5 + 0.8, and 4.4 ~lt0.8 kg, respectively. In three other cats, we increased the dose of intrathecal MEN 10.207 to 500 pg. Contraction-induced reflex increases in MAP and HR before and after 500 hg MEN 10.207 were identical, 41 ?z 15 mmHg and 2 t 2 beats/min and 41 + 15 mmHg and 2 + 2 beats/min, respectively. 240
15r
2
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2
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120
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2. Summary data for cardiovascular and ventilatory responses of 7 cats to static contraction of triceps surae muscles before (filled columns) and 16 min after (hatched columns) CP 96,345 (means + SE). Reflex changes in MAP and minute volume of ventilation (VI) are significantly attenuated after CP 96,345. Reflex change in HR is not significantly attenuated. * Significantly different (P < 0.05). FIG.
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PRESSOR REFLEX 250
T
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=
225
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200
: e
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-5 150 z 125 ” too z 75 4 50 E 25 0 n=6
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FIG. 3. Effects of intrathecal injection of 100 rg [D-Pro2,D-Phe7,D-Trp’]SP (D-Pro) on reflex cardiovascular and ventilatory responses to static contraction before (closed columns), 10 min after (hatched columns), and >30 min after (open columns) ~-Pro (means 2 SE). Reflex change in MAP is significantly attenuated after ~-Pro. More than 30 min later, reflex change in MAP has returned to level achieved before D-Pro. Reflex changes in HR and %‘I are not significantly attenuated after D-Pro. * Significantly different (P < 0.05).
Before MEN 10.207 the ventilatory response to contraction was 279 + 62 ml/min, whereas after 500 pg MEN 10.207 this response was 222 2 60 ml/min (P > 0.05). The pressor and ventilatory responses to static contraction were significantly attenuated (P < 0.05) after intrathecal lidocaine (17 f 3 mmHg and 53 f 3 ml/min, respectively). Before MEN 10.207, after MEN 10.207, and after lidocaine, the developed tensions of the triceps surae muscles averaged 6.1 f 0.6, 5.6 + 1.1, and 5.7 + 0.8 kg, respectively.
NK-3 antagonist. In five cats the reflex pressor, chronotropic, and ventilatory responses to static contraction of the triceps surae muscles were measured before and 10 min after the intrathecal injection of the NK-3 antagonist Nle”. Before Nle”, static contraction evoked increases in MAP, HR, and VI of 19 f 1 mmHg, 12 + 2 beats/min, and 106 + 42 ml/min, respectively. After Nlel’, reflex increases in MAP, HR, and VI averaged 24 + 4 mmHg, 14 + 3 beats/min, and 203 + 37 ml/min, respectively. Contraction-induced reflex increases in MAP, HR, and VI before Nlel’ and after Nle” were not
significantly different (P > 0.05). The developed tensions of the triceps surae muscles averaged 5.6 + 1 and 5.6 -t 1 kg. DISCUSSION
We have shown that the injection of the selective NK-1 receptor antagonist CP 96,345 into the cerebrospinal fluid bathing the lumbosacral
spinal cord attenuated
the
reflex pressor and ventilatory responses to static contraction of the triceps surae muscles. In addition, we confirmed a previous finding that the reflex pressor response to static muscular contraction is attenuated by [DPro’,D-Phe7,D-Trpg]SP, an NK-l/NK-2 receptor antagonist (12). We were unable to provide evidence that NK-2 or NK-3 receptors are involved in the spinal transmission of the exercise pressor reflex. We do not believe that the attenuating effects of CP 96,345 on the exercise pressor reflex can be explained by a neurotoxic effect of this antagonist. McLean et al. (17) showed that the application of CP 96,345 to the medium perfusing locus ceruleus neurons did not alter their baseline firing rates. In addition, we do not believe that our
results are a consequence of the effect of CP 96,345 on sympathetic outflow. Using Evans blue dye as an index of the extent of spread of an injectate,
we showed that the
thoracic spinal segments, in which sympathetic outflow to the heart and lungs originates, were not stained blue after the intrathecal
injection
of Evans blue dye onto
the lumbosacral spinal cord. Moreover, we previously showed that sympathetic outflow, as assessedby the cardiovascular
and ventilatory
responses either to electrical
stimulation of sciatic nerve afferents synapsing in the upper lumbar cord (7-9) or to electrical stimulation of the posterior hypothalamus (13) was not affected by substances injected onto the lower lumbar and sacral spinal cord. A role for NKA in the spinal transmission of the exercise pressor reflex has yet to be defined. Recently, NKA
was detected in the dorsal horn of the spinal cord after repeated isometric contractions of the feline hindlimb muscles (4). This finding suggested to us that NKA may play a neurotransmitter/modulatory
role in the exercise
pressor reflex. Our attempts to demonstrate such a role were unsuccessful. The reflex cardiovascular and ventilatory
responses to a static muscular
contraction
were
unaffected after the intrathecal injection of the NK-2 receptor antagonist MEN 10.207. This lack of effect by MEN 10.207 on the exercise pressor reflex probably was not a consequence of the inaccessibility of MEN 10.207 to the appropriate spinal laminae of the dorsal horn, because
[D-Pro2,D-Phe7,D-Trp’]SP
or lidocaine,
which
were injected intrathecally through the same cannula and in the same volume as MEN 10.207, attenuated the reflex responses to static contraction.
Nevertheless,
we
cannot exclude the possibility that MEN 10.207 was ineffective because of incomplete blockade of NK-2 receptors in the dorsal horn. Although
we injected intrathe-
tally lOO- and 500-pg doses of MEN 10.207, a sufficient number of NK-2 receptors may have remained available for NKA activation. We speculate that NKA functioning as an endogenous ligand for NK-2 receptors in the dorsal horn does not participate in the spinal transmission of the exercise pressor reflex. In our experiments, only NK-1 receptor blockade, with either the specific NK-1 receptor antago-
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THE
nist or the combined NK-l/NK-2 receptor antagonist, attenuated the pressor and ventilatory responses to contraction. If NKA is a spinal neuromodulator of the exercise pressor reflex, NKA probably exerts its effects through the SP receptor NK-1. Endogenous NKA has been reported to be a ligand for the NK-1 and NK-2 receptors (23). The functional significance for the existence of NK-3 receptors in the dorsal horn is yet to be elucidated. Conflicting evidence has been presented concerning the involvement of NK-3 receptors in the spinal processing of somatosensory information. For example, Laneuville et al. (14) found that intrathecal NKB increased reaction time to a radiant heat stimulus. Conversely, FleetwoodWalker et al. (5) found that the responses of spinal cervical tract neurons to noxious stimuli were unaffected after the microiontophoretic application of either a NK-3 receptor agonist, Senkitide, or antagonist, Nle”. We, too, were unable to identify a physiological role for spinal NK-3 receptors, hence, our finding that the cardiovascular and ventilatory responses to static muscular contraction were unaffected after the intrathecal injection of the NK-3 receptor antagonist Nlel’. SP has been identified as a putative neurotransmitter or neuromodulator of contraction-activated afferent input to the dorsal horn of the spinal cord. Two pieces of evidence point to neuromodulation rather than to neurotransmission as the role played by SP. First, SP has been shown to induce a slow depolarization of dorsal horn neurons (25). Slow depolarization is characteristic of a modulator of afferent input to the dorsal horn, whereas rapid synaptic action is characteristic of a transmitter (2). Second, exogenous SP microinjected into the feline L, dorsal horn with the hindlimb muscles at rest has been shown to have no effect on blood pressure or HR (28). If SP had acted as a neurotransmitter, the cardiovascular changes that occur with static exercise should have been mimicked by the application of SP into the dorsal horn. We have tested three hypotheses in an attempt to identify a role for each of three related tachykinins, SP, NKA, and NKB, in the spinal transmission of the exercise pressor reflex. Using NK-2 (NKA) and NK-3 (NKB) receptor antagonists, we were unable to show any change in the cardiovascular or ventilatory responses to static muscular contraction. Using an NK-1 (SP) receptor antagonist, we were able to attenuate the pressor and ventilatory responses to static muscular contraction. Our findings are good evidence linking SP and NK-1 receptors with the exercise pressor reflex. We speculate that the role played by SP is one of spinal neuromodulation of this reflex. We thank Charles Stuart for technical assistance. We also thank Pfizer, Inc., for the generous gift of CP 96,345. This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-30710. J. M. Hill was supported by American Heart Association, California Affiliate, Fellowship 90-17. J. G. Pickar was supported by NHLBI National Research Service Award HL-08144. Address for reprint requests: J. M. Hill, Div. of Cardiovascular Medicine, TB172, University of California, Davis, CA 95616. Received
16 December
1991; accepted
in final
form
28 April
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23. 24.
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