Regulatory Peptides, 29 (1990) 199-213

199

Elsevier REGPEP 00933

Tonic enhancement of the sensitivity of baroreceptor reflex response by endogenous substance P in the rat Julie Y . H . Chart 1, Charles D. Barnes 3 and Samuel H . H . C h a n 2 ~Department of Medical Research, Taipei Veterans General Hospital, 21nstitute of Pharmacology, National Yang-Ming Medical College, Taipei, Taiwan (Republic of China) and 3Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University,Pullman, WA (U.S.A.)

(Received 11 December 1989;revised version received and accepted 12 April 1990) K e y words: Substance P; (D-Pro 2, D-Trp7'9)-substance P; Substance P antiserum;

Bestatin; Phosphoramidon; Baroreceptor reflex sensitivity; Nucleus tractus solitarius; Rat

Summary Modulation of baroreceptor reflex (BRR) by endogenous substance P (SP) in the brain was investigated in rats anesthetized with pentobarbital sodium. Intracerebroventricular administration of the undecapeptide (15 or 30 nmol) and its antagonist, (D-Pro 2, D-Trp7'9)-SP (30 or 60 nmol) or SP antiserum (1 : 20), respectively, promoted a significant increase and decrease in the sensitivity of BRR response. Prolonging the endogenous activity of SP with the aminopeptidase blocker, bestatin (200 nmol) or with the endopeptidase-24.11 inhibitor, phosphoramidon (200 nmol) significantly augmented the same reflex. Combining the undecapeptide with either peptidase blocker, moreover, promoted additional potentiation of the BRR response. On the other hand, simultaneous administration of bestatin and (D-Pro 2, D-WrpT"9)-SPproduced a reduction of the augmented effect of bestatin on the sensitivity of BRR response. Bilateral microinjection of SP (600 pmol) or an antiserum against SP (1 : 20) into the nucleus tractus solitarius (NTS) elicited respectively an enhancement of and reduction in the BRR response. These data suggest that neurons that contain SP may participate in central cardiovascular control by tonically enhancing the sensitivity of the BRR response, possibly via an action on the NTS.

Correspondence: S. H. H. Chart, Institute of Pharmacology,National Yang-MingMedical College,Taipei

11221, Taiwan, Republic of China. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

200 Introduction

Substance P (SP) is a peptide that was originally extracted from equine brain and intestine in the form of a dry powder, and was named preparation P by von Euler and Gaddum [i]. It was first partially [2], and later completely [3], purified from bovine hypothalamus and characterized as an undecapeptide. In addition to causing intestinal contraction and lowering blood pressure via vasodilatation [ 1], this naturally occurring undecapeptide is traditionally linked to the transmission of pain signals in the spinal cord [4]. Over the past decade, however, SP has gained interest as a blood pressureregulating neuropeptide. Based on results from immunocytochemical, autoradiographic, neurochemical and electrophysiologic studies, SP is believed to participate in circulatory control in at least two fashions. First, a bulbospinal SP pathway [5-9], which originates in the ventral surface of the medulla oblongata and terminates on the intermediolateral cell column of the thoracic spinal cord, is a major pathway involved in the regulation of vasomotor sympathetic outflow [ 10]. Second, SP is a putative neurotransmitter or neuromodulator at the baroreceptor afferent terminals located in the nucleus tractus solitarius (NTS) [11-14]. Although many studies have been carried out to investigate the effects of directly microinjecting SP into the NTS on basal arterial pressure and heart rate [ 11,15-17], relatively little is known of the action of the undecapeptide on the baroreceptor reflex (BRR) control system, the sensitivity of which is crucial to the maintenance of a steady arterial pressure. Whereas exogenous application of SP is the most logical approach in initial investigations, the data obtained only concern its potential action on the BRR response. They do not, however, prove that the endogenous undecapeptide actually possesses such a property. Furthermore, the results may be pharmacologic rather than physiologic. Evaluation of the endogenous activity of SP, on the other hand, would establish the suggested biologic function and demonstrate the existence of tonic, intrinsic activity. Such an evaluation, however, is hindered by the relatively low concentration and short half-life of the undecapeptide in the central nervous system. Several peptidases capable of hydrolyzing SP have been identified in the brain, and their activities can be blocked by selective inhibitors [18-22]. These degradative enzyme blockers (prolong endogenous activity), together with selective SP antagonists or antiserum against SP (block intrinsic activity), offer an excellent opportunity to investigate the endogenous action of this neuropeptide. The present study employed a recently developed peptide treatment scheme from our laboratory [23,24], and exploited the properties of two aminopeptidase inhibitors [ 18-22,25,26], bestatin (arginyl-aminopeptidase inhibitor) and phosphoramidon (endopeptidase-24.11 inhibitor), a SP antagonist, (D-Pro 2, D-Trp7'9)-SP [27-29] and an antiserum against SP, to examine the action of endogenous SP on the sensitivity of BRR response. Additionally, the possibility that the NTS, where baroreceptor afferents terminate [ 11,30], may be a site of action for SP, was investigated. Together, these experiments suggest that neurons that contain SP may exert a tonic enhancement on the sensitivity of the BRR response, possibly via an action on the NTS.

201 Materials and Methods

General preparation Experiments were carried out in adult, male Sprague-Dawley rats (245-296 g) anesthetized with pentobarbital sodium (40 mg/kg, i.p., with 10 mg/kg/h, i.v. supplements). Intubation of the trachea and cannulation of the right femoral artery and vein were routinely performed to facilitate ventilation, measure systemic arterial pressure and administer drugs. The head of the animal was thereafter securely fixed in a stereotaxic apparatus (Kopf). Body temperature was maintained at 37 °C through a thermostatically controlled heating system. The pulsatile and mean systemic arterial pressure (SAP and MSAP), as well as heart rate (HR, monitored via a cardiotachometer triggered by the arterial pulses), were continuously displayed on a Grass polygraph.

Intracerebroventricular (i.c. v.) administration Following the procedure used in previous studies [23,24,31], a 25-gauge stainlesssteel cannula was implanted into the lateral cerebral ventricle as the guide cannula. The coordinates for the lateral ventricle were 1.3-1.5 mm from the midline, 0.8-1.0 mm posterior to the bregma, and 4.5-5.0 mm from the skull surface. I.c.v. injection of SP, (D-Pro 2, D-Trp7"9)-SP, SP antiserum, bestatin, phosphoramidon, artificial cerebrospinal fluid (aCSF) or normal rabbit serum was carried out by inserting a 27-gauge, flatly bevelled needle, into the guide cannula. The former was connected to a 10-/~l Hamilton microliter syringe by a PE-20 polyethylene tubing. A total volume of 2.5/A was delivered over at least 1 min to allow for full diffusion of the solution.

Microinjection of peptides Direct microinjection of SP or SP antiserum into the NTS was carried out, using a nanoliter infusion pump (WPI) and through a glass micropipette. A total volume of 20 nl was delivered over 2 min into the NTS on each side. Direct injection of aCSF or normal rabbit serum, at the same volume and infusion rate, served as the respective vehicle control. To aid in histologic verification of microinjection sites, 19/0 Evans blue was added to the injecting medium.

Measurement of the sensitivity of baroreceptor reflex response The sensitivity of BRR response was assessed by a modification of the method of Smythe et al. [32]. In essence, we measured the maximal reflex reduction in heart rate in response to transient increases in arterial pressure produced by different doses of phenylephrine (2.5, 5.0 or 10.0 ~tg/kg, i.v.). The slope of the regression line that relates these two circulatory parameters was used as the index for the sensitivity of the BRR response. To avoid sequential dependency on arterial pressure changes, and hence the BRR responses, the three doses of phenylephrine were injected in a random manner.

Experimental protocol Since SP has been reported to affect the respiratory control system [ 33 ], animals were artificially ventilated with a Harvard respirator throughout the recording session. Our first series of experiments examined the action of applying SP (30 nmol), (D-Pro 2,

202 D-Trp7,9)-SP (60 nmol), bestatin (200 nmol), phosphoramidon (200 nmol) or aCSF to the lateral ventricle on basal SAP and HR (n = 4 animals per group). The time-course effect of SP (15 or 30 nmol), (D-Pro 2, D-Trp7"9)-SP (30 or 60 nmol) or SP antiserum (1:20) on the sensitivity of BRR response was investigated in the second set of experiments (n = 6 animals per group). The slope of the regression relationship was examined before, and at 5-15, 25-35, and 55-65 min (note that it takes approximately 10 min to complete the induction of 3 B RR responses) after administration of peptides into the lateral ventricle. In our third set of experiments, the effect of bestatin (200 nmol, i.c.v.) on changes in the sensitivity of BRR response induced by SP (30 nmol, i.c.v.) was studied by concomitantly administering the degradative enzyme inhibitor with the undecapeptide (n = 5 animals per group). This procedure was repeated in a fourth set of experiments (n = 5 animals per group), employing phosphoramidon and SP (200 and 30 nmol, i.c.v.). Our fifth set of experiments (n = 6 animals per group) evaluated the effect of simultaneous i.c.v, administration ofbestatin (200 nmol) and (D-Pro 2, D-Trp7,9)-SP (30 or 60 nmol). The action of bilateral microinjection of SP (600 pmol), aCSF, antiserum against SP (1 : 20) or normal rabbit serum (1 : 20) into the NTS on the sensitivity of BRR response (n = 6 animals per group) or basal SAP and HR (n = 4 animals per group) was studied in our final set of experiments. Drugs Phenylephrine (Sigma) was freshly made up with saline, and SP (Sigma), (D-Pro 2, D-Trp7"9)-SP (Sigma), bestatin (Sigma), phosphoramidon (Sigma), SP antiserum (Immuno Nuclear) or normal rabbit serum (Jackson) thawed, immediately before use. I.c.v. injection of aCSF or normal rabbit serum served as the vehicle control. Histology The brain was removed after each experiment and fixed in 30yo sucrose-10~ formaldehyde-saline solution for at least 48 h. Histologic verifications of the position ofi.c.v. injection or microinjection sites were carried out on frozen 25-/~m sections stained with Cresyl violet. Statistics The effects of various treatments on the sensitivity of BRR response were statistically assessed using two-way analysis of variance with repeated measurements, followed by the Student-Newman-Keuls test for a posteriori multiple comparisons at corresponding time intervals.

Results

Effects of SP, (D-Pro2, D-Trp7"9)-SP,bestatin and phosphoramidon on basal arterial pressure and heart rate Table I summarizes the respective effect of various peptide agents used in this study on basal SAP and HR. I.c.v. administration of SP (30 nmol) elicited hypotension and

203 TABLE I Effects ofi.c.v, administration of SP, (D-Pro 2, D-TrpT'9)-SP, bestatin and phosphoramidon on basal arterial pressure and heart rate Values are m e a n + S.E.M. (n = 4 animals per group).

SP (30 nmol) (D-Pro 2, D-Trp7,9)-SP (60 nmol) Bestatin (200 nmol) Phosphoramidon (200 nmol) aCSF

Mean systemic arterial pressure

Heart rate

maximal change (mmHg)

duration (min)

maximal change (bpm)

duration (min)

- 18.3 + 2.1

23.3 + 8.2

+41.0 + 5.6

24.5 + 0.7

+ 17.5 + 3.5 + 25.3 + 2.5

30.0 _+ 7.1 > 60

- 12.5 + 3.5 + 43.4 + 4.6

6.5 + 0.7 > 60

+ 7.8 + 0.9 + 6.8 +_ 2.2

> 60 56.7 + 8.5

+ 5.8 _+ 0.7 + 7.7 + 1.4

> 60 > 60

tachycardia that endured more than 20 min. Its antagonist (60 nmol), on the other hand, produced hypertension and bradycardia, with the former effect outlasting the latter by more than 20 min. Bestatin (200 nmol) induced its reported [31] increase, whereas phosphoramidon (200 nmol) did not promote any significant alteration, in SAP and HR. Time-course effects of SP, its antagonist or SP antiserum on the sensitivity of baroreceptor reflex response I.c.v. administration of S P (15 or 30 nmol) elicited a significant enhancement of the sensitivity of BRR response (Fig. 1). This augrnentatory action of the undecapeptide followed a time course that maximized at 25-35 min postinjection, and was still appreciable at 55-65 min with the higher dose.

i ~SP~15r~ tnr~ 2.o-

[ ] v~bc~

1.5

lwtao~:

:

0.5

0

, control 5-15 25-35 55-65 ~POSTINJECTION TIME (rain)

Fig. 1. Time-course changes in baroreceptor reflex sensitivity slopes following i.c.v, administration of SP (15 or 30 nmol) or aCSF (vehicle). Values presented are m e a n + S.E.M. (n = 6 animals per group). *P < 0.05 and **P < 0.01 vs. aCSF at comparable time points in the Student-Newman-Keuls analysis.

204

t.5"r-

Vehicle SP Antagonist (30 rm~O

• sP An~mc~t (6o nmoO

E

E O.

[] []

1.0"

e~ UJ a.

00.5112 nm

i

i

i

i

control

5-15

25-35

55-65

POSTINJECTION TIME (min)

Fig. 2. Time-course changes in baroreceptor reflex sensitivity slopes following i.c.v, administration of (D-Pro 2, D-Trp7"9)-SP (30 or 60 nmol) or aCSF (vehicle). Values presented are mean + S.E.M. (n = 6 animals per group). *P < 0.05 vs. aCSF in the Student-Newman-Keuls analysis.

To confirm that the central augmentatory effect of SP on the BRR response was not a consequence of peripheral leakage, we examined the systemic action of the undecapeptide in another group of animals. We found that compared to saline treatment, intravenous administration of SP (30 nmol) elicited no apparent change in the sensitivity of BRR response (BRR slope: 0.82 + 0.12 vs 0.77 + 0.08 bpm/mmHg, mean + S.E.M., n = 4) at 25-35 min postinjection, the time period during which i.c.v. SP at the same dose maximally increased the sensitivity of the same reflex (Fig. 1). The SP antagonist, (D-Pro 2, D-TrpT'9)-SP (30 nmol), when applied via the i.c.v, route (Fig. 2), showed a general trend, though statistically insignificant, of reduction in the sensitivity of the BRR. This depressive effect became discernible at 25-35 min postinjection when a higher dose of the antagonist was employed. The tonic facilitatory action of endogenous SP on BRR response implied by treatment with its antagonist was further assessed using an antiserum against SP raised in rabbit. Compared to normal rabbit serum (1 : 20), application of SP antiserum (1 : 20) into the lateral ventricle also produced a significant (P < 0.01, n = 4) reduction in the sensitivity of BRR response (BRR slope: 0.68 + 0.03 vs 0.38 + 0.04 bpm/mmHg, mean + S.E.M., n = 4) that maximized at 25-35 min postinjection.

Effects of simultaneous administration of SP and bestatin or phosphoramidon on the sensitivity of baroreceptor reflex response I.c.v. application of the arginyl-aminopeptidase inhibitor, bestatin (200 nmol), as reported previously [23,24,31], significantly increased the sensitivity of the BRR response (Fig. 3). This augmentative effect was further potentiated when SP (15 nmol) was administered simultaneously with bestatin (200nmol). Phosphoramidon (200 nmol, i.c.v.), the endopeptidase-24.11 inhibitor that purportedly prolongs the efficacy of endogenous SP, produced a similar effect. Its enhancing action on the sensitivity of BRR response was further augmented (Fig. 4) when it was injected (200 nmol) together with SP (15 nmol).

205 ~ 60 ~

~ 60" E

Y

z 30

< =Z

o Vehicle Bestatin '~ SP + Bestattn

[

!

z 30

¢,r

i

I

0

2.5 5.0 10.0 PHENYLEPHRINE (pg/kg, Lv.~ 0 2.5 5.0 10.0

Z

I

30"

*

I

Vehicle t~Phosphoramidon z~ SP + Phosphoramidon o