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Immunoneutralization of substance P attenuates the reflex pressor response to muscular contraction
Brain Research, 377 (1986) 199-203 Elsevier
199
BRE 21640
Immunoneutralization of substance P attenuates the reflex pressor response to muscular contraction MARC P. KAUFMAN, KENNETH J. RYBICKI, GERALD P. KOZLOWSKI, and GARY A. IWAMOTO Departments of Physiology and Cell Biology and Anatomy, and the Harry S. Moss Heart Center, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, TX 75235 (U. S. A.)
(Accepted March 18th, 1986) Key words: antiserum - - exercise - - reflex cardiovascular control - - spinal cord - - muscle afferent
We previously reported that subarachnoid injection of a peptide antagonist to substance P attenuated by half the reflex pressor response to static muscular contraction. Subsequently, some of the peptide antagonists to substance P have been found to possess local anesthetic effects, Therefore, we have repeated our experiments using a substance P antiserum, which was shown to be without local anesthetic effect. We found that intrathecal injection of the antiserum attenuated by more than half the reflex pressor response to static contraction of the triceps surae muscles of cats.
Static contraction of hindlimb muscles in anesthetized cats has been well established to reflexly increase arterial pressure, heart rate and myocardial contractility2,9,1°. F u r t h e r m o r e , group III and IV muscle afferents have been shown to comprise the afferent limb of the reflex arc causing these cardiovascular responses to contraction 1°. A l t h o u g h the neurotransmitters and n e u r o m o d u l a t o r s r e l e a s e d by group II1 and IV afferents are not p r o v e n , substance P is clearly one candidate. This p e p t i d e has been found in the terminals of fine caliber axons running in the L 6$1 dorsal roots 11. In addition, electrical stimulation of A 6 - and C-fiber afferents in the sciatic nerve has been shown to release substance P in the spinal cord 11. A l s o , i o n t o p h o r e t i c application of substance P has been shown to stimulate dorsal horn cells receiving somatic afferent input 3. Recently, we d e m o n s t r a t e d that injection of the substance P antagonist, D-Pro 2, D-Phe 7, D-Trp9-sub stance P~ into the s u b a r a c h n o i d space a t t e n u a t e d by half the reflex pressor response to static muscular contraction 6. Subsequently, however, the selectivity of peptide antagonists for substance P has been challenged by Karlsson et al. 5, who found that m a n y of
them had local anesthetic effects. This local anesthetic effect might have been responsible for the attenuation by the substance P antagonist of the reflex pressor response to static contraction observed in our previous r e p o r t 6. W e therefore sought to re-examine the role played by substance P in causing the reflex pressor response to static muscular contraction. Instead of using a peptide antagonist to substance P, we used the antiserum to this peptide, an agent whose chemical structure is very different from that of the p e p t i d e antagonist believed to cause local anesthesia. W e found that the ' i m m u n o n e u t r a l i z a t i o n ' of substance P within the spinal cord m a r k e d l y a t t e n u a t e d the reflex pressor response to static muscular contraction. Ten cats, weighing 2 . 5 - 4 . 5 kg, were anesthetized with a-chloralose (80 mg/kg; i.p.). The right jugular vein, right carotid artery and cervical trachea were cannulated. The l u m b a r and sacral spinal cord were exposed and a PE-50 catheter was introduced into the subarachnoid space so that its tip was placed about 1 cm rostral to the entry point into the spinal cord of the L 7 dorsal root. This subarachnoid cannula, which had a dead space of 0.2 ml, was secured
Correspondence: M.P. Kaufman, Department of Physiology, University of Texas, Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235, U.S.A.
200 with a purse string suture (6-0) placed in the dura mater. Arterial pressure was measured by connecting the carotid cannula to a Statham P23ID transducer. H e a r t rate was calculated beat-to-beat from the arterial pulse with a G o u l d biotachometer. Tension developed by the contraction of the triceps surae muscles was measured by attaching the calcaneal tendon to a force transducer (Grass, FT-10). A r t e r i a l pressure, heart rate, and tension were written on a Hewlett-Packard 7404A chart recorder. The lungs were ventilated with r o o m air by a H a r v a r d respirator. Arterial blood gases were measured periodically and were maintained within normal limits. The effect of injecting the substance P antiserum (1 ml) into the subarachnoid cannula on the reflex pressor response to static contraction of the triceps surae muscles was examined. The antiserum was generated in rabbits and had a titer of 1:12,500, which represents the dilution that binds 50% of a standard amount of antigen. The amount of binding was determined by radioimmunoassay. In addition, the antiserum did not cross-react (i.e. less than 0.01%) with neurotensin, M e t - e n k e p h a l i n a m i d e or somatostatin. It did, however, cross-react with physalaemin, an amphibian peptide, and with the peptide fragment, substance p4-11. The triceps surae muscles were statically contracted for 30-45 s by electrically stimulating the tibial nerve at 40 Hz (pulse duration = 25 kts; two times m o t o r threshold). The time between contractions was at least 20 min. Injection of 1 ml of n o r m a l rabbit serum into the subdural cannula had no effect on the pressor response to static contraction in three cats. Likewise injection of 1 ml of substance P antisera, absorbed with 1 mg of substance P, had no effect on the pressor response to contraction in two cats. M o r e over, injection of 1 mt of an oxytocin antibody had no effect on this response to contraction in two cats. All values are r e p o r t e d as mean + S . E . M . Analysis of variance followed by Scheff6 post hoc tests was used to determine statistical significance. We found that administration of the substance P antiserum significantly attenuated the pressor responses to static contraction in 9 of the 10 cats tested (Figs. 1, 2A). On the average, contraction before giving the antiserum increased m e a n arterial pressure by 27 + 6 mm Hg ( P < 0.01). Contraction,
15-20 min after injecting the substance P antiserum, increased mean arterial pressure by only 11 + 2 m m Hg, an effect which was significantly above control levels ( P < 0.01) but which was less than half the increase e v o k e d by the previous contraction ( P < 0.01). Contraction of the triceps surae 7 0 - 9 0 min after injecting the antiserum resulted in a partial restoration of the pressor response. Thus mean arterial pressure increased above control levels by 22 + 4 mm Hg, an effect which was significantly smaller than the increase e v o k e d by the initial contraction ( P < 0.01) but which was significantly greater than the increase evoked by the second contraction ( P < 0.01). We also found that injection of the antiserum attenuated the heart rate increase e v o k e d by static contraction in 9 of the 10 cats tested (Fig. 2B). On the average, contraction before giving the antiserum increased heart rate by 17 + 3 b p m above control levels (P < 0.01). Contraction after giving the antiserum increased heart rate by 11 + 3 bpm, an effect which was significantly above control levels (P < 0.05), but
150
( mm Hg )
A
B
c
D
--
50 --
HR
t80 --
(bpm)
90 -5--
~
Tension (kg)
O---150
(mrn Hg)
HR ( bpm )
Tension (kg)
50
- -
,j,,.,=i,i
--
180 -90 --
5 --
~ - ~
0----
Fig. 1. Effects of antisera to substance P and to ovalbumin on the reflex pressor response to static contraction. A: static contraction of the triceps surae muscles reflexly increased arterial pressure and heart rate. B: the reflex pressor response to static contraction was unaffected by subarachnoid injection of ovalbumin antisera. C: subarachnoid injection of substance P antisera attenuated the reflex pressor response to static contraction. D: 70 min after subarachnoid injection of substance P antisera the pressor response to static contraction was partially restored.
201 A. I
150 140 MAP (ram Hg)
150
ii l
120 110
100
B.
I
210 200
2.
HR 190 (bpm) t80 17o
~1 ~!
*
-
F..L::!:
I
........ii .. i~i~ii '3:'2.':~
C 40Hz Before
z C 40Hz After After (15-20rain) (70-90min)
Fig. 2. Summary of the reflex pressor-tachycardia responses to static contraction of the triceps surae muscles before, 15-20 min after and 70-90 min after subarachnoid injection of substance P antiserum. Asterisks denote statistical significance (P < 0.01) between control (C) and peak responses to contraction (40 I-Iz). Bars and their associated vertical brackets represent means and standard errors, respectively. Horizontal brackets placed at the top of A and B connect groups whose pressor (A) tachycardic (B) responses to contraction were significantly different (P < 0.01) from each other.
which was significantly less than the increase evoked by the first contraction (P < 0.01). Contraction 70-90 min after giving the antiserum did not result in a significant restoration of the tachycardic response to contraction. Thus heart rate increased by 12 + 3 bpm, an effect which was significantly above control levels (P < 0.01) but which was not significantly different from that evoked by the second contraction (P > 0.05). The peak tensions developed by the three contractions were not significantly different from each other (P > 0.05) and averaged 3.9 + 0.4, 4.0 + 0.4 and 3.7 + 0.4 kg, respectively. In 3 of the 10 cats, we determined the extent of
spread of the antiserum injection. To accomplish this we injected 1 ml of Evans Blue dye into the spinal cannula. We found that 15 min after its injection, the dye spread from the sacral to cervical regions of the spinal cord. Because of the extensive spread, we needed to control for the possibility that the substance P antiserum attenuated the pressor response to contraction by inhibiting sympathetic outflow at the thoracic and lumbar levels of the spinal cord. Therefore, in 6 of the 10 cats, we examined the effect of the antiserum injection on the pressor response evoked by electrical stimulation (80 Hz; 0.5 ms; 240MA for 30 s) of the posterior hypothalamic area. We found that subdural injection of the antiserum had no effect on either the arterial pressure or heart rate responses to hypothalamic stimulation (Fig. 3). For example, mean arterial pressure increased by 27 + 4 mm Hg before antiserum injection and by 30 + 8 mm Hg after injection. In 3 of the 10 cats, we examined the effect of injecting into the subarachnoid cannula 1 ml of ovalbumin antisera obtained from goat (Arnel Products). We found that injection of the ovalbumin antisera had no effect on either the increase in mean arterial pressure or the increase in heart rate evoked by static contraction of the triceps surae muscles. Last, we paralyzed each of the 10 cats with decamethonium bromide (0.3 mg/kg; i.v.) and stimulated the tibial nerve with the same parameters as those which both statically contracted the triceps surae and which reflexly increased mean arterial pressure and heart rate. Stimulation of the tibial nerve in paralyzed cats had no effect on either heart rate or arterial pressure. In two paralyzed cats which did not participate in the reflex studies, we determined the local anesthetic properties of the substance P antiserum. To accomplish this, we recorded the compound action potential from the I_,7 dorsal root while we placed caudal to the recording site a 15 mm strip of filter paper soaked in the antiserum. The compound action potential, which was evoked by single pulse stimulation of the tibial nerve, showed waves indicative of activation of groups I through IV afferents. These potentials were unaffected by the antiserum. However, a similar strip of filter paper soaked in 1% lidocaine markedly attenuated the group IV wave of the compound action potential.
202 A. 160 "150
MAP (mm Hg)
140 "150 "120 "1"10 "100
200 "190 HR (bpm)
"180 470 "160
C Stim Before
C Stirn After
Fig. 3. Summary of the effect of stimulation of the posterior hypothalamus on mean arterial pressure (A) and heart rate (B) before and after subarachnoid injection of substance P antiserum. Asterisks denote statistical significance (P < 0.025) between control (C) and peak responses to hypothalamic stimulation (Stim). Bars and their associated vertical brackets represent means and standard errors, respectively. Note that the pressor response evoked by stimulation before antiserum injection was not significantly different from that evoked by stimulation after antiserum injection.
Our results have shown that immunoneutralization with a substance P antiserum injected into the cerebrospinal fluid bathing the lumbosacral spinal cord attenuated by more than half the reflex pressor response to static contraction of the triceps surae muscles. The origin of this attenuation was unlikely to have been at the level of the sympathetic outflow because injection of the antiserum did not affect the pressor responses to posterior hypothalamic stimulation. In addition, the attenuation was unlikely to have been due to a non-specific immunoglobulin effect because injection of either ovalbumin antiserum or absorbed substance P antiserum had no effect on
the reflex cardiovascular responses to static contraction. As shown in Figs. 1 and 2A, injection of the substance P antiserum increased control mean arterial pressure. Although we cannot explain the mechanism causing this increase, the effect might be used to explain the attenuation of the reflex pressor response to contraction. This explanation, however, is not supported by our finding that a similar antiserum-induced increase in control arterial pressure did not attenuate the pressor response to hypothalamic stimulation (Fig. 3A). At first glance our finding that the substance P antiserum had no effect on the pressor response evoked by hypothalamic stimulation might appear to conflict with the findings of Loewy and Sawyer s and Keeler and Helke 7, who reported that chemical activation of the ventrolateral medulla produced a pressor response that was greatly attenuated by intrathecal injection of substance P antagonists. It is important to recognize, however, that in our experiments the tip of the spinal cannula was located at L 7 and was pointing caudally, conditions intended to minimize the access of the antiserum to the thoracolumbar sympathetic outflow. By contrast, the tip of the spinal cannula in the experiments of Loewy and Sawyer 8 and Keeler and Helke 7 was located at the thoracic level in an attempt to maximize access of their substance P antagonists to the sympathetic outflow. Furthermore, it is not known whether the pathway from the posterior hypothalamus to the sympathetic preganglionic cell body involves substance P. With these considerations in mind, our experiments, while useful in demonstrating a role for substance P in the reflex arc increasing arterial pressure during muscular contraction, shed little light on the role of this peptide in controlling synaptic events in the intermediolateral cell column during activation of pressor sites in the medulla. Using immunoneutralization as a means of preventing the spinal actions of substance P, we have confirmed our previous report 6 which used a peptide antagonist to demonstrate a role for substance P in the reflex pressor response to static contraction. The fact that the reflex pressor response was attenuated but not abolished by the substance P antiserum may have been due to substance P release overwhelming the binding capacity of the antiserum. Alternatively,
203 other substances such as somatostatin 4 may play important roles in the spinal transmission of afferent input from working muscle. We thank Mr. James Jones for his expert technical assistance and Ms. Janet Wright and Ms. Pamela De
1 Bj6rkroth, U., Rosell, S., Xu, J.-C. and Folkers, K., Pharmacological characterization of four related substance P antagonist, Acta Physiol. Scand., 116 (1982) 167-173. 2 Coote, J.H., Hilton, S.M. and Perez-Gonzalez, J.F., The reflex nature of the pressor response to muscular exercise, J. Physiol. (London), 215 (1971) 780-804. 3 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 4 H6kfelt, T., Elde, R., Johansson, R., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 5 Karlsson, J.A., Finney, M.J.B., Persson, C.G.A. and Post, C., Substance P antagonists and the role of tachykinins in non-cholinergic bronchoconstriction, Life Sci., 35 (1984) 2681-2691. 6 Kaufman, M.P., Kozlowski, G.P. and Rybicki, K.J., Attenuation of the reflex pressor response to muscular contraction by a substance P antagonist, Brain Research, 333
Roxtra for preparing the manuscript. Supported by N I H Grants HL06296 and HL30710, a G r a n t - i n Aid from the A m e r i c a n Heart Association (83-1179), and the Lawson and Rogers Lacy Research F u n d in Cardiovascular Diseases. M.P.K. is an Established Investigator of the A m e r i c a n Heart Association.
(1985) 182-184. 7 Keeler, J.R. and Helke, C.J., Spinal cord substance P mediates bicuculline-induced activation of cardiovascular responses from the ventral medulla, J. Auton. Nerv. Syst., 13 (1985) 19-33. 8 Loewy, A.D. and Sawyer, W.B., Substance P antagonist inhibits vasomotor responses elicited from ventral medulla in rat, Brain Research, 245 (1982) 379-383. 9 McCloskey, D.I. and Mitchell, J.H., Reflex cardiovascular and respiratory responses originating in exercising muscle, J. Physiol. (London), 224 (1972) 173-186. 10 Mitchell, J.H., Reardon, W.C. and McCloskey, D.I., Reflex effects on circulation and respiration from contracting skeletal muscle, Am. J. Physiol., 233 (1977) H374-H378. 11 Pickel, V.M., Reis, D.J. and Leeman, S.E., Ultrastructural localization of substance P in neurons of rat spinal cord, Brain Research, 122 (1977) 534-540. 12 Yaksh, T.L., Jessell, T.M., Gamse, R., Mudge, A.W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord, in vivo, Nature (London), 286 (1980) 155-157.
199
BRE 21640
Immunoneutralization of substance P attenuates the reflex pressor response to muscular contraction MARC P. KAUFMAN, KENNETH J. RYBICKI, GERALD P. KOZLOWSKI, and GARY A. IWAMOTO Departments of Physiology and Cell Biology and Anatomy, and the Harry S. Moss Heart Center, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, TX 75235 (U. S. A.)
(Accepted March 18th, 1986) Key words: antiserum - - exercise - - reflex cardiovascular control - - spinal cord - - muscle afferent
We previously reported that subarachnoid injection of a peptide antagonist to substance P attenuated by half the reflex pressor response to static muscular contraction. Subsequently, some of the peptide antagonists to substance P have been found to possess local anesthetic effects, Therefore, we have repeated our experiments using a substance P antiserum, which was shown to be without local anesthetic effect. We found that intrathecal injection of the antiserum attenuated by more than half the reflex pressor response to static contraction of the triceps surae muscles of cats.
Static contraction of hindlimb muscles in anesthetized cats has been well established to reflexly increase arterial pressure, heart rate and myocardial contractility2,9,1°. F u r t h e r m o r e , group III and IV muscle afferents have been shown to comprise the afferent limb of the reflex arc causing these cardiovascular responses to contraction 1°. A l t h o u g h the neurotransmitters and n e u r o m o d u l a t o r s r e l e a s e d by group II1 and IV afferents are not p r o v e n , substance P is clearly one candidate. This p e p t i d e has been found in the terminals of fine caliber axons running in the L 6$1 dorsal roots 11. In addition, electrical stimulation of A 6 - and C-fiber afferents in the sciatic nerve has been shown to release substance P in the spinal cord 11. A l s o , i o n t o p h o r e t i c application of substance P has been shown to stimulate dorsal horn cells receiving somatic afferent input 3. Recently, we d e m o n s t r a t e d that injection of the substance P antagonist, D-Pro 2, D-Phe 7, D-Trp9-sub stance P~ into the s u b a r a c h n o i d space a t t e n u a t e d by half the reflex pressor response to static muscular contraction 6. Subsequently, however, the selectivity of peptide antagonists for substance P has been challenged by Karlsson et al. 5, who found that m a n y of
them had local anesthetic effects. This local anesthetic effect might have been responsible for the attenuation by the substance P antagonist of the reflex pressor response to static contraction observed in our previous r e p o r t 6. W e therefore sought to re-examine the role played by substance P in causing the reflex pressor response to static muscular contraction. Instead of using a peptide antagonist to substance P, we used the antiserum to this peptide, an agent whose chemical structure is very different from that of the p e p t i d e antagonist believed to cause local anesthesia. W e found that the ' i m m u n o n e u t r a l i z a t i o n ' of substance P within the spinal cord m a r k e d l y a t t e n u a t e d the reflex pressor response to static muscular contraction. Ten cats, weighing 2 . 5 - 4 . 5 kg, were anesthetized with a-chloralose (80 mg/kg; i.p.). The right jugular vein, right carotid artery and cervical trachea were cannulated. The l u m b a r and sacral spinal cord were exposed and a PE-50 catheter was introduced into the subarachnoid space so that its tip was placed about 1 cm rostral to the entry point into the spinal cord of the L 7 dorsal root. This subarachnoid cannula, which had a dead space of 0.2 ml, was secured
Correspondence: M.P. Kaufman, Department of Physiology, University of Texas, Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235, U.S.A.
200 with a purse string suture (6-0) placed in the dura mater. Arterial pressure was measured by connecting the carotid cannula to a Statham P23ID transducer. H e a r t rate was calculated beat-to-beat from the arterial pulse with a G o u l d biotachometer. Tension developed by the contraction of the triceps surae muscles was measured by attaching the calcaneal tendon to a force transducer (Grass, FT-10). A r t e r i a l pressure, heart rate, and tension were written on a Hewlett-Packard 7404A chart recorder. The lungs were ventilated with r o o m air by a H a r v a r d respirator. Arterial blood gases were measured periodically and were maintained within normal limits. The effect of injecting the substance P antiserum (1 ml) into the subarachnoid cannula on the reflex pressor response to static contraction of the triceps surae muscles was examined. The antiserum was generated in rabbits and had a titer of 1:12,500, which represents the dilution that binds 50% of a standard amount of antigen. The amount of binding was determined by radioimmunoassay. In addition, the antiserum did not cross-react (i.e. less than 0.01%) with neurotensin, M e t - e n k e p h a l i n a m i d e or somatostatin. It did, however, cross-react with physalaemin, an amphibian peptide, and with the peptide fragment, substance p4-11. The triceps surae muscles were statically contracted for 30-45 s by electrically stimulating the tibial nerve at 40 Hz (pulse duration = 25 kts; two times m o t o r threshold). The time between contractions was at least 20 min. Injection of 1 ml of n o r m a l rabbit serum into the subdural cannula had no effect on the pressor response to static contraction in three cats. Likewise injection of 1 ml of substance P antisera, absorbed with 1 mg of substance P, had no effect on the pressor response to contraction in two cats. M o r e over, injection of 1 mt of an oxytocin antibody had no effect on this response to contraction in two cats. All values are r e p o r t e d as mean + S . E . M . Analysis of variance followed by Scheff6 post hoc tests was used to determine statistical significance. We found that administration of the substance P antiserum significantly attenuated the pressor responses to static contraction in 9 of the 10 cats tested (Figs. 1, 2A). On the average, contraction before giving the antiserum increased m e a n arterial pressure by 27 + 6 mm Hg ( P < 0.01). Contraction,
15-20 min after injecting the substance P antiserum, increased mean arterial pressure by only 11 + 2 m m Hg, an effect which was significantly above control levels ( P < 0.01) but which was less than half the increase e v o k e d by the previous contraction ( P < 0.01). Contraction of the triceps surae 7 0 - 9 0 min after injecting the antiserum resulted in a partial restoration of the pressor response. Thus mean arterial pressure increased above control levels by 22 + 4 mm Hg, an effect which was significantly smaller than the increase e v o k e d by the initial contraction ( P < 0.01) but which was significantly greater than the increase evoked by the second contraction ( P < 0.01). We also found that injection of the antiserum attenuated the heart rate increase e v o k e d by static contraction in 9 of the 10 cats tested (Fig. 2B). On the average, contraction before giving the antiserum increased heart rate by 17 + 3 b p m above control levels (P < 0.01). Contraction after giving the antiserum increased heart rate by 11 + 3 bpm, an effect which was significantly above control levels (P < 0.05), but
150
( mm Hg )
A
B
c
D
--
50 --
HR
t80 --
(bpm)
90 -5--
~
Tension (kg)
O---150
(mrn Hg)
HR ( bpm )
Tension (kg)
50
- -
,j,,.,=i,i
--
180 -90 --
5 --
~ - ~
0----
Fig. 1. Effects of antisera to substance P and to ovalbumin on the reflex pressor response to static contraction. A: static contraction of the triceps surae muscles reflexly increased arterial pressure and heart rate. B: the reflex pressor response to static contraction was unaffected by subarachnoid injection of ovalbumin antisera. C: subarachnoid injection of substance P antisera attenuated the reflex pressor response to static contraction. D: 70 min after subarachnoid injection of substance P antisera the pressor response to static contraction was partially restored.
201 A. I
150 140 MAP (ram Hg)
150
ii l
120 110
100
B.
I
210 200
2.
HR 190 (bpm) t80 17o
~1 ~!
*
-
F..L::!:
I
........ii .. i~i~ii '3:'2.':~
C 40Hz Before
z C 40Hz After After (15-20rain) (70-90min)
Fig. 2. Summary of the reflex pressor-tachycardia responses to static contraction of the triceps surae muscles before, 15-20 min after and 70-90 min after subarachnoid injection of substance P antiserum. Asterisks denote statistical significance (P < 0.01) between control (C) and peak responses to contraction (40 I-Iz). Bars and their associated vertical brackets represent means and standard errors, respectively. Horizontal brackets placed at the top of A and B connect groups whose pressor (A) tachycardic (B) responses to contraction were significantly different (P < 0.01) from each other.
which was significantly less than the increase evoked by the first contraction (P < 0.01). Contraction 70-90 min after giving the antiserum did not result in a significant restoration of the tachycardic response to contraction. Thus heart rate increased by 12 + 3 bpm, an effect which was significantly above control levels (P < 0.01) but which was not significantly different from that evoked by the second contraction (P > 0.05). The peak tensions developed by the three contractions were not significantly different from each other (P > 0.05) and averaged 3.9 + 0.4, 4.0 + 0.4 and 3.7 + 0.4 kg, respectively. In 3 of the 10 cats, we determined the extent of
spread of the antiserum injection. To accomplish this we injected 1 ml of Evans Blue dye into the spinal cannula. We found that 15 min after its injection, the dye spread from the sacral to cervical regions of the spinal cord. Because of the extensive spread, we needed to control for the possibility that the substance P antiserum attenuated the pressor response to contraction by inhibiting sympathetic outflow at the thoracic and lumbar levels of the spinal cord. Therefore, in 6 of the 10 cats, we examined the effect of the antiserum injection on the pressor response evoked by electrical stimulation (80 Hz; 0.5 ms; 240MA for 30 s) of the posterior hypothalamic area. We found that subdural injection of the antiserum had no effect on either the arterial pressure or heart rate responses to hypothalamic stimulation (Fig. 3). For example, mean arterial pressure increased by 27 + 4 mm Hg before antiserum injection and by 30 + 8 mm Hg after injection. In 3 of the 10 cats, we examined the effect of injecting into the subarachnoid cannula 1 ml of ovalbumin antisera obtained from goat (Arnel Products). We found that injection of the ovalbumin antisera had no effect on either the increase in mean arterial pressure or the increase in heart rate evoked by static contraction of the triceps surae muscles. Last, we paralyzed each of the 10 cats with decamethonium bromide (0.3 mg/kg; i.v.) and stimulated the tibial nerve with the same parameters as those which both statically contracted the triceps surae and which reflexly increased mean arterial pressure and heart rate. Stimulation of the tibial nerve in paralyzed cats had no effect on either heart rate or arterial pressure. In two paralyzed cats which did not participate in the reflex studies, we determined the local anesthetic properties of the substance P antiserum. To accomplish this, we recorded the compound action potential from the I_,7 dorsal root while we placed caudal to the recording site a 15 mm strip of filter paper soaked in the antiserum. The compound action potential, which was evoked by single pulse stimulation of the tibial nerve, showed waves indicative of activation of groups I through IV afferents. These potentials were unaffected by the antiserum. However, a similar strip of filter paper soaked in 1% lidocaine markedly attenuated the group IV wave of the compound action potential.
202 A. 160 "150
MAP (mm Hg)
140 "150 "120 "1"10 "100
200 "190 HR (bpm)
"180 470 "160
C Stim Before
C Stirn After
Fig. 3. Summary of the effect of stimulation of the posterior hypothalamus on mean arterial pressure (A) and heart rate (B) before and after subarachnoid injection of substance P antiserum. Asterisks denote statistical significance (P < 0.025) between control (C) and peak responses to hypothalamic stimulation (Stim). Bars and their associated vertical brackets represent means and standard errors, respectively. Note that the pressor response evoked by stimulation before antiserum injection was not significantly different from that evoked by stimulation after antiserum injection.
Our results have shown that immunoneutralization with a substance P antiserum injected into the cerebrospinal fluid bathing the lumbosacral spinal cord attenuated by more than half the reflex pressor response to static contraction of the triceps surae muscles. The origin of this attenuation was unlikely to have been at the level of the sympathetic outflow because injection of the antiserum did not affect the pressor responses to posterior hypothalamic stimulation. In addition, the attenuation was unlikely to have been due to a non-specific immunoglobulin effect because injection of either ovalbumin antiserum or absorbed substance P antiserum had no effect on
the reflex cardiovascular responses to static contraction. As shown in Figs. 1 and 2A, injection of the substance P antiserum increased control mean arterial pressure. Although we cannot explain the mechanism causing this increase, the effect might be used to explain the attenuation of the reflex pressor response to contraction. This explanation, however, is not supported by our finding that a similar antiserum-induced increase in control arterial pressure did not attenuate the pressor response to hypothalamic stimulation (Fig. 3A). At first glance our finding that the substance P antiserum had no effect on the pressor response evoked by hypothalamic stimulation might appear to conflict with the findings of Loewy and Sawyer s and Keeler and Helke 7, who reported that chemical activation of the ventrolateral medulla produced a pressor response that was greatly attenuated by intrathecal injection of substance P antagonists. It is important to recognize, however, that in our experiments the tip of the spinal cannula was located at L 7 and was pointing caudally, conditions intended to minimize the access of the antiserum to the thoracolumbar sympathetic outflow. By contrast, the tip of the spinal cannula in the experiments of Loewy and Sawyer 8 and Keeler and Helke 7 was located at the thoracic level in an attempt to maximize access of their substance P antagonists to the sympathetic outflow. Furthermore, it is not known whether the pathway from the posterior hypothalamus to the sympathetic preganglionic cell body involves substance P. With these considerations in mind, our experiments, while useful in demonstrating a role for substance P in the reflex arc increasing arterial pressure during muscular contraction, shed little light on the role of this peptide in controlling synaptic events in the intermediolateral cell column during activation of pressor sites in the medulla. Using immunoneutralization as a means of preventing the spinal actions of substance P, we have confirmed our previous report 6 which used a peptide antagonist to demonstrate a role for substance P in the reflex pressor response to static contraction. The fact that the reflex pressor response was attenuated but not abolished by the substance P antiserum may have been due to substance P release overwhelming the binding capacity of the antiserum. Alternatively,
203 other substances such as somatostatin 4 may play important roles in the spinal transmission of afferent input from working muscle. We thank Mr. James Jones for his expert technical assistance and Ms. Janet Wright and Ms. Pamela De
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Roxtra for preparing the manuscript. Supported by N I H Grants HL06296 and HL30710, a G r a n t - i n Aid from the A m e r i c a n Heart Association (83-1179), and the Lawson and Rogers Lacy Research F u n d in Cardiovascular Diseases. M.P.K. is an Established Investigator of the A m e r i c a n Heart Association.
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