Regulatory Peptides, 28 (1990) 23-37

23

Elsevier REGPEP 00886

Tonic suppression of baroreceptor reflex by endogenous neurotensin in the rat Chiung-Tong Chen

1,

Julie Y . H . Chan ~,2, Charles D. Barnes 2 and Samuel H . H . Chan !

~Depanment and Institute of Pharmacology, National Yang-MingMedical College, Taipei, Taiwan (Republic of China) and 2Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA (U.S.A.)

(Received 19 June 1989;revisedversion received28 October 1989; accepted 7 November 1989) K e y words: Neurotensin; (D-Trpl ~)_neurotensin; Neurotensin antiserum; Bestatin;

Baroreceptor reflex sensitivity; Nucleus tractus solitarius; Rat

Summary We evaluated the modulatory role of endogenous neurotensin (NT) in baroreceptor reflex (BRR) response in Sprague-Dawley rats anesthetized with pentobarbital sodium. Intracerebroventricular (i.c.v.) administration of NT (15 or 30 nmol) significantly reduced the sensitivity of the BRR response. Blocking the endogenous activity of the tridecapeptide with its specific antagonist, (o-Trpll)-NT (4 or 8 nmol) or antiserum against NT (1 : 20); or inhibiting the aminopeptidases with bestatin (200 nmol), on the other hand, promoted a potentiation of BRR response. When administered together with bestatin (200 nmol), the suppressive effect of NT (15 nmol) on the BRR response was further enhanced, as was the augmentative action of(D-Trpll)-NT (4 nmol). Upon microinjection into the bilateral nucleus tractus solitarius (NTS), NT (600 pmol) and (D-Trp 1~)-NT (150 pmol) respectively elicited a reduction and enhancement of the B RR response. These results suggest that neurons that contain NT may participate in central cardiovascular regulation by tonically suppressing the BRR, possibly via an action on the NTS where baroreceptor afferents terminate.

Correspondence: S.H.H. Chan, Institute of Pharmacology,National Yang-Ming Medical College,Talpei

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

24 Introduction

Neurotensin (NT) is a tridecapeptide that was isolated and characterized by Carraway and Leeman [1] in 1973 from bovine hypothalamic extracts. Apart from eliciting hypotension, from which it derived its name [ 1], NT is now known to participate in many other physiologic functions [e.g., 2-6], including hypothermia, nociception, glucoregulation, gastric secretion and gonadotropin release. Most of the earlier work [ 1,7-9] on the cardiovascular effects of NT were directed towards its peripheral actions. Results from immunocytochemical, autoradiographic and receptor binding studies [ 10-14], however, suggested the likely engagement of the tridecapeptide in the central regulatory machinery for circulatory functions. Rioux et al. [ 15] first reported in 1981 that intracerebroventricular administration of NT in conscious and pentobarbital-anesthetized rats promotes a dose-dependent hypotension. Other authors [16-18], however, observed an increase in arterial pressure and/or a decrease in renin activity in conscious rats. Apart from these equivocal results, little is known of the hemodynamic effects of centrally-applied NT, in particular, the baroreceptor reflex (BRR), the sensitivity of which is crucial to the maintenance of a steady arterial pressure. As the most logical initial step, studies using exogenous application of NT only concern the potential biologic function(s) of this neuropeptide. They do not, however, prove that the tridecapeptide actually possesses such an intrinsic property. Furthermore, the results obtained may be pharmacologic rather than physiologic. Evaluation of the endogenous activity of NT, on the other hand, would establish the suggested biologic function and demonstrate the existence of tonic, intrinsic activity. The present study utilized an experimental scheme that we recently developed to evaluate the endogenous functions of neuropeptides [19]. We exploited the properties of bestatin, an aminopeptidase inhibitor that potentially protects NT from catabolism [20-22]; and (D-Trp~I)-NT, the selective NT antagonist [23,24] and an antiserum against NT, both of which block the tonic, intrinsic activity of the tridecapeptide, to evaluate the participation of endogenous NT in the modulation of BRR response. Additionally, the possibility that the nucleus tractus solitarius (NTS), where baroreceptor afferents terminate [10,25], may be a site of action for NT, was investigated. Together, these experiments suggest that neurons that contain NT may exert a tonic inhibitory action on the BRR, possibly via an action on the NTS.

Materials and Methods

General preparation Adult, male Sprague-Dawley rats (216-270g), anesthetized with pentobarbital sodium (50 mg/kg, i.p., with 10 mg/kg per h i.v. supplements), were used in this study. The trachea in each animal was intubated to facilitate ventilation, and the right femoral artery and vein were cannulated for the measurement of systemic arterial pressure and introduction of drugs. Pulsatile and mean systemic arterial pressure (SAP and MSAP), and heart rate (HR) monitored via a cardiotachometer triggered by the arterial pulses, were routinely displayed on a Grass polygraph.

25

lntracerebroventricular (i.c. v.) injection Following the procedures used in a previous study [26], a 25-gauge stainless-steel cannula was implanted into the lateral cerebral ventricle on the right side at (mm): P 0.8-1.0, R 1.4-1.7 and H 3.5-4.5 with reference to the bregma. I.c.v. injection of NT, (D-Trp~I)-NT, anti-NT, bestatin or artificial cerebrospinal fluid (aCSF)was carried out by inserting a 27-gauge, flatly bevelled needle, into the guide cannula. The former was connected to a 10 #I Hamilton microliter syringe by a PE-20 polyethylene tubing. A total volume of 5 #1 was delivered over at least 1 min to allow for full diffusion of the solution.

Microinjection of peptides Direct microinjection of NT or (D-Trp~)-NT into the NTS was carried out, using a nanoliter infusion pump and through a glass micropipette. A total volume of 20 nl was delivered into the bilateral NTS over 2 min. Direct injection ofaCSF at the same volume and infusion rate served as the vehicle control,

Evaluation of baroreceptor reflex response The sensitivity of baroreceptor reflex (BRR) response was evaluated in two different fashions. The first (ratio method) [ 19,27] utilized a quotient that represents the unit change in reflex bradycardia per unit increase in MSAP (b/rain per mmHg) elicited by a single dose of phenylephrine (5 #g/kg, i.v.). Individual values were subsequently normalized to a percent of control to compensate for variations between animals. The second (slope method) is a modification of the method reported by Smythe et al. [28]. In essence, we employed the slope of the regression line that relates the maximal reflex reduction in HR to elevations in SAP elicited by three doses of phenylephrine (2.5, 5 and 10 #g/kg, i.v.). The order of different doses of phenylephrine was altered randomly to avoid sequential dependency.

Experimental procedures The temporal effects following i.c.v, administration of NT (15 or 30nmol), (D-Trp~I)-NT (4 or 8 nmol), or bestatin (200nmol) on basal SAP and HR were evaluated in our first series of experiments. Our second series of experiments investigated the time-course action of various peptide treatments on the sensitivity of BRR response, using the ratio method. In these experiments, the BRR was induced before, and during the first 5 min of every 10-rain interval over 60-65 min following i.c.v. administration of NT (15 or 30nmol), (D-TrpI1)-NT (4 or 8nmol), NT plus (D-Trpl~)-NT (15 and 4 nmol), anti-NT (1:20), bestatin (200 nmol), NT plus bestatin (15 and 200 nmol), or (D-Trp ~~)-NT plus bestatin (4 and 200 nmol). Examination of the sensitivity of BRR response, utilizing the slope method, was carded out in our third series of experiments. The effects of most of the above peptide treatments were studied, however, during the postinjection time when they exhibited peak activity, as revealed in the time-course evaluation from our second series of experiments. The temporal effects following bilateral microinjection into the NTS of NT (600 pmol), (D-Trp ~l)-NT (150 pmol) or aCSF on the sensitivity of BRR response were evaluated in the fourth series of experiments, using the ratio method, at 10-min intervals during the 60-65 min postinjection.

26

NT (Sigma), (D-Trp I 1)-NT (Sigma) or bestatin (Sigma) was freshly prepared with aCSF, phenylephrine (Sigma) with saline, and rabbit antiserum against NT (Immune Nuclear) thawed, immediately before use. I.c.v. injection of aCSF served as the vehicle control in all experiments, with the exception of anti-NT, which employed normal rabbit serum as the vehicle control.

Histology The brain was removed after each experiment and f'lxed in 30% sucrose/10% formaldehyde-saline solution for at least 48 h. Histologic verifications of the position of i.c.v, injection or microinjection sites were carried out on frozen 25-/~m sections stained with Cresyl violet. Identification of microinjection sites in the NTS was aided by the addition of 1 ~/o Evans blue in the injection medium. Statistics

The effects ofpeptide treatments on the sensitivity of BRR response were statistically assessed using two-way analysis of variance (ANOVA) with repeated measurements. This was followed by the Student-Newman-Keuls test for a posteriori multiple comparisons at corresponding time intervals to determine the significance of their temporal actions. A

•

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Fig. 1. Time-course alterations in basal MSAP and HR following i.c.v, administration of aCSF (Q), NT (15 nmol, II) or NT (30nmol, &) (A); and aCSF (O), (D-Trp")~NT (4nmol, l l ) or (D-TrpII)-NT (8 nmol, &) (B). The initial MSAP and HR of each group in A were, respectively, 107 + 4 and 410 + 15, 108 + 4 and 400 + 20, and 101 + 2 and 397 + 11; and in B 101 + 2 and 394 + 12, 103 + 5 and 396 + 16, and 101 + 2 and 397 + 11 (mmHg and b/rain). Values presented are mean + S.E.M. (n = 8 animals per group). * P < 0,05 and * * P < 0.01 vs. aCSF at comparable time-points in the Student-Newman-Keuls test.

27

Results Temporal effects of neurotensin, (D- Trp 1~)-neurotensin or bestatin on basal arterial pressure and heart rate I.c.v. administration of both doses of N T (15 or 30 nmol) which we employed produced a minor and transient hypertension. These were followed by hypotension and bradycardia (Fig. 1A), in which the degree and duration were dependent on the dose. The lower dose of N T (15 nmol) reduced SAP and H R that reached their peaks at 10-15 min postinjection; whereas the same effects by the higher dose (30nmol) maximized 20-25 min following i.c.v, injection. (D-Trp ~ )-NT (4 nmol, i.c.v.) did not elicit an appreciable alteration in SAP, although it produced a reduction in H R (Fig. 1B) that became significantly (P < 0.05, n = 8) different from control during 20-55 min postinjection. I.c.v. administration of a higher dose of the N T antagonist (8 nmol), on the other hand, produced appreciable (P < 0.05 or 0.01, n = 8) hypotension and bradycardia that reached their respective peaks at 25 and 35 min (Fig. 1B). As revealed in previous experiments [19,27], bestatin (200 nmol, i.c.v.) promoted hypertension and tachycardia (peak increase: 25.4 + 2.3 mmHg, and 43.7 + 4.5 b/min, mean + S.E.M., n = 8). Temporal effects of neurotensin, or its antagonist or antiserum on the baroreceptor reflex response I.c.v. administration of N T (15 or 30 nmol) elicited a reduction in the sensitivity of BRR response (Fig. 2). This depressant action of the tridecapeptide followed a timecourse that was only significantly (P < 0.01, n --- 7) different from vehicle control during the initial 15 min. Since both doses of N T were only appreciably (P < 0.05, n = 7)

20

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Fig. 2. Time-coursechangesin the sensitivityof BRR responsefollowingi.c.v,administrationofaCSF (open bar), NT (15 nmol, stippled bar), or NT (30 nmol, solid bar). The initial MSAP and HR of each group were respectively 105 _+4 and 390 _+10, 104 _+3 and 381 _+ 11, and 103 _+3 and 398 _+8 (mmHg and b/min). Values presented are mean _+S.E.M. (n = 7 animals per group). **P < 0.01 vs. aCSF at comparable time-points in the Student-Newman-Keulstest.

28

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POSTINJECTION TIME (rain) Fig. 3. Time-course changes in the sensitivity ofBRRresponse foUowingi.c.v, admirtistration ofaCSF (open bar), (D-Trp tt )-NT (4 nrnol, stippled bar), or (D-Trp I l )-NT (8 nmol, solid bar). The initial M S A P and HR of each group were respectively 108 + 3 and 371 + 9, 105 + 7 and 413 + 10, and 103 + 3 and 398 + 8 (mmHg and b/rain). Values presented are mean + S.E.M. (n = 7) animals per group). *P < 0.05 and • * P < 0.01 vs. aCSF at comparable time-points in the Student-Newman-Keuls test.

different from each other at 5 min postinjection, the lower dose (15 nmol) was used throughout the remainder of this study. The NT antagonist, (D-Trp ll)-NT (4 nmol), when applied via the i.c.v, route (Fig. 3), produced an enhancement of the sensitivity of BRR response that was appreciably (P < 0.05, n --- 7) different from the action of aCSF at 10-25 min postinjection. This potentiating effect on the BRR response became augmented, both in degree and duration (Fig. 3), upon a higher dose of the NT antagonist (8 nmol). Although some early reports [23,24] identified (D-Trp~I)-NT as an active antagonist for NT in peripheral tissues, the same receptor antagonistic effect following its central administration has not been characterized. The latter effect was demonstrated in the present study. Rather than producing a suppressive action, simultaneous i.c.v, administration of NT and (D-Trp I ])-NT (15 and 4 nmol) elicited an enhancement of the BRR response that reached a peak of + 47.4 + 5.3% (mean + S.E.M., n = 4). Since 4 nmol of (D-Trp~I)-NT was capable of antagonizing the exogenously applied (15 nmol) NT, this dose was employed in subsequent experiments. The tonic inhibitory action of endogenous NT on BRR response implied by treatment with its antagonist was further assessed using a rabbit antiserum against NT. Whereas i.c.v, administration of normal rabbit serum (1 : 20) produced minimal effect on the BRR response (peak effect: - 3.4 + 6.5%, mean + S.E.M., n = 4), antiserum against NT (1 : 20) elicited a significant (P < 0.05, n = 4) augrnentatory action on the same reflex, with a peak effect of + 39.6 + 1.8% (mean + S.E.M., n = 4).

Temporal effects of combined administration of bestatin and neurotensin on the baroreceptor reflex response I.c.v. application of bestatin (200 nmol), as we reported previously [ 19,27], significantly increased the sensitivity of the BRR that maximized at 40-45 rain postinjection

29

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Fig. 4. Time-course changes in the sensitivityof BRR response following i.c.v, administration of bestatin (200 nmol, open bar) or bestatin plus NT (200 and i 5 nmol, solid bar). Data on NT (15 nmol, stippled bar) from Fig. 2 were redrawn for comparison.The initial MSAP and HR of each group were respectively105 + 6 and 386 + 10, 101 + 4 and 400 + 9, and 105 + 4 and 390 ± 10 (mmHg and b/rain). Values presented are mean ± S.E.M. (n = 7 animals per group). *P < 0.05 and **P < 0.01 vs. NT (15 nmol); Ip < 0.05 and sp < 0.01 vs. bestatin at comparable time-points in the Student-Newman-Keulstest. (Fig. 4). Administering N T (15 nmol) simultaneously with bestatin (200 nmol), however, resulted in a reversal of the augmentative effect of the aminopeptidase inhibitor on the BRR response (Fig. 4), to an inhibitory action that was significantly (P < 0.05 or 0.01, n = 7) different from bestatin treatment during 10-35 min postinjection. The results produced by bestatin administration, we reason, represent the net action on the sensitivity of B R R response of all the endogenous neuropeptides (including NT) that are potentially protected by this aminopeptidase inhibitor [21,22,29]. Superimposed upon this action should logically be the additional effect of the specifically heightened exogenous activity of NT, when it was given simultaneously with bestatin. It follows that the specifically enhanced action of exogenously applied N T by the aminopeptidase on the sensitivity of BRR can be revealed by: (bestatin + N T ) (bestatin) at comparable time intervals. Fig. 5 demonstrates the outcome of such a mathematical treatment. Significant augmentation (P < 0.05 or 0.01, n = 7), both in degree and duration, of the depressant effect of N T (15 nmol) on BRR response become very apparent when the tridecapeptide was administered simultaneously with bestatin.

Temporal effects of combined administration of bestatin with neurotensin antagonist on the baroreceptor reflex response Based on similar reasoning, we also determined the net action on B R R response of all the endogenous neuropeptides that are potentially protected by bestatin, minus the contribution of endogenous NT. This was provided by simultaneous i.c.v, administration of bestatin (200 nmol) and (D-TrpI1)-NT (4 nmol). Such a treatment signifi-

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c a n t l y ( P < 0.01, n = 7) p o t e n t i a t e d the e n h a n c i n g effect o f the N T a n t a g o n i s t o n the sensitivity o f B R R r e s p o n s e (Fig. 6). A s u b t r a c t i o n o f these results f r o m t h o s e o b t a i n e d with b e s t a t i n t r e a t m e n t alone (Fig. 7) revealed specifically the h e i g h t e n e d s u p p r e s s a n t a c t i o n o f e n d o g e n o u s N T o n the B R R r e s p o n s e , w h i c h is a m i r r o r i m a g e o f the t e m p o r a l effect o f (D-Trp 11)-NT (4 n m o l ) .

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POSTINJECTION TIME (min)

Fig. 7. Mathematical management of data from Fig. 6 to demonstrate the time-course of specifically enhanced effects (stippled bar) of endogenous NT on the sensitivity of BRR response by bestatin (200 nmol i.c.v.). Data on (D-TrpH)-NT (4 nmol, i.c.v., open bar) from Fig. 3 were redrawn for comparison.

Effects of peptide treatments on phenylephrine-induced hypertension Since the transient hypertension induced by phenylephrine, as called for by our protocol, had to be superimposed upon a basefine arterial pressure already modified by the peptide treatments, our results may have been confounded because the degree of increase in SAP was compromised. That this may not be the case is shown in Table I. Phenylephrine, for example, elicited similar amplitudes of vasopression before, and at various time intervals, after i.c.v, administration of NT (15 nmol), (D-Trpll)-NT (4 nmol), bestatin (200 nmol) or aCSF. Thus, it appears that the reflex alterations in HR in response to elevations of SAP evoked at different time periods following peptide administrations reasonably reflect the temporal effects of these treatments on the BRR response. Nonetheless, as a further precaution, we specifically defined the BRR response as a quotient representing the unit change in HR per unit change in MSAP (b/min per mmHg).

TABLE I

The degree of hypertension (mmHg) induced by phenylephrine (5 #g/kg, i.v.) before and at various postinjection intervals following different i.c.v, peptide treatments: aCSF, NT (15 nmol), (D-Trpn)-NT (4 nmol) or bestatin (200 nmol) Values presented are mean ± S.E.M. (n = 7 animals per group). Before aCSF NT (D-Trpal)-NT Bestatin

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10-15 rain

20-25 rain

30-35 rain

37.3 35.9 34.4 32.8

37.8 36.9 31.5 32.3

37.3 38.5 32.2 32.3

37.8 38.5 30.0 32.2

+ ± ± ±

2.3 2.8 9.4 3.3

+ 2.3 ± 2.8 _+ 8.1 ± 2.8

+ + ± ±

2.2 2.5 6.6 2.8

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2.2 2.5 4.2 2.9

32 A

B

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TREATMENT Fig. 8. Effects of various i.c.v, peptide treatments on the sensitivity of BRR response, as evaluated by the slope method. The initial MSAP and HR of each group (A, B, C, D, E, F) were respectively 106 + 2 and 408 5: 12, 108 + 3 and 408 + 5, 110 + 5 and 409 + 13, 99 + 2 and 388 + 16, 108 + 5 and 410 + 13, and 106 + 4 and 390 5:14 (mmHg and b/rain). Values presented are mean + S.E.M. (n = 6 animals per group). *P < 0.05, **P < 0.01 vs. aCSF, t p < 0.05 vs. bestatin in the ANOVA analysis.

Effects of peptide treatments on the sensitivity of baroreceptor reflex response as determined by the slope method The effects on the sensitivity of BRR response of various peptides were determined during the postinjection time interval when they exhibited peak activity. Thus, evaluation of the BRR response, using the slope method, was carried out before, and 80

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POSTINJECTION TIME (min) Fig. 9. Time-course changes in the sensitivity of the BRR response following bilateral microinjection of aCSF (open bar), NT (60tl p~ol, stippled bar), or (D-Trptl)-NT (150 pmol, solid bar) into the NTS. The initial M S A P and HR of each group were 100 + 5 and 369 + 13, 104 + 5 and 374 5: 19, and 102 + 5 and 379 + 16 (mmHg and b/rain). Values presented are mean + 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 test.

33 A

[3

Fig. 10. Diagrammatic representations of transverse sections through two levels of the caudal medulla showing the microinjectionsites of NT and (D-TrpI~)-NTin the NTS. Abbreviations:AP, area postrema; NA, nucleus ambiguus, NC, nucleus cuneatus; ND, subnucleus reticularis dorsalis medullae oblongatae; NG, nucleus gracilis; NOI, nucleus olivarisinferior;NR, nucleus raphe; NRL, nucleus reticularislateralis; NTS, nucleus tractus solitarius; NV, subnucleus reticularis ventralis medullae oblongatae; PY, tractus pyramidalis; PYX, deeussatiopyramidium;TS, tractus solitarius; V, nucleus and tractus trigeminispinalis; X, nucleus dorsalis nerve vagi; XII, nucleus hypoglossi. 10-15 min following N T (15 nmol), (D-Trp11)-NT (4 nmol) or aCSF, or 40-45 min after bestatin (200 nmol) was delivered. Fig. 8 summarizes the results from this series of experiments. As expected, N T (15 nmol) and (D-Trp~l)-NT (4 nmol) promoted, respectively, a significant (P < 0.05 or 0.01, n = 6) reduction and enhancement of the sensitivity of BRR response. Likewise, bestatin (200 nmol) increased the slope of BRR response, which was appreciably (P < 0.05, n -- 6) reduced when the aminopeptidase inhibitor was administered simultaneously with N T (15 nmol), but was further augmented when bestatin was delivered together with (D-Trpl~)-NT (4 nmol).

Time-course effects of microinjection of neurotensin or its antagonist into the NTS on the baroreceptor reflex response Fig. 9 demonstrates that suppression of the BRR response by the endogenous N T may take place at the NTS. In comparison to the aCSF, microinjection of N T (600 pmol) directly into the NTS elicited a marked (P < 0.01, n = 6) reduction in the sensitivity of the BRR response. On the other hand, a blockade of the endogenous activity of N T with (D-Trp 11)-NT (150 pmol), applied via the same route, produced a significant (P < 0.01, n --- 6) enhancement of the same reflex. Both the magnitude and duration of the suppressive action of the tridecapeptide, and the enhancing effect of its antagonist, were augmented when compared to those produced by the same peptides upon i.c.v, injection (cf. Figs. 2, 3). Histologically, the distribution of verified injection sites was primarily concentrated in the dorsomedial region of the NTS (Fig. 10), where a high density of N T nerve terminals and receptor binding sites has been demonstrated [ 10,13], and where baroreceptor afferents terminate [ 10]. Discussion The present study demonstrated that neurons that contain N T may participate in central cardiovascular regulation by tonically reducing the sensitivity of BRR response via an action on the NTS. We demonstrated that i.c.v, application of N T suppressed

34 and that (D-Trp~)-NT, antiserum against NT and bestatin potentiated the BRR response. Furthermore, when administered simultaneously with bestatin, the suppressive effect of NT on the BRR response was further enhanced, as was the augmentative action of (D-Trp ~ )-NT. At the nuclear level, direct microinjection of NT into the NTS decreased, whereas its receptor antagonist increased, the BRR response. As we pointed out previously [ 19,27], and again demonstrated in this sttidy, it is highly likely that the modulatory action of NT on the BRR is exerted in concert with other brain peptides that may also play a role in this important cardiovascular regulatory process. Based on its properties as a blocker of arginyl-, leucine-, alanyl- and leucylaminopeptidase [21,22, 29], bestatin may potentially modulate the BRR response by enhancing the tonic endogenous activities of at least AIII, neurotensin, substance P and somatostatin in the brain [27]. Thus, the facilitation of bestatin on the BRR may conceivably represent the net action of all these neuropeptides, and may give way to a suppression when the balance was tilted towards NT upon its exogenous administration. Inhibiting the endogenous activity of NT with (D-Trp 11)-NT, it follows, further increased the augmentative effect of bestatin on the BRR response. We are aware that the interpretation of our results is limited by the specificity of the various peptide agents employed. As demonstrated in the present study, (D-Trp H)-NT was capable of antagonizing the central effect of NT, and its effect on the BRR response was similar to that of the antiserum against NT. The limitation of specificity is also minimized by the treatment scheme that we devised for the present study, which exploits the properties of, and is based on, an interplay between NT, (D-Trp ~)-NT and bestatin. The mathematical management of our data also allowed us to reveal the specifically heightened exogenous and endogenous activity of NT (cf. Figs. 5, 7). It is interesting to note that whereas NT has a rather short half-life [20], its effect on the BRR response endured for more than 20 min. One possible explanation is that the action of the tridecapeptide may involve the participation of second messengers. I.c.v. administration of NT produces in the hypothalamus a respective decrease and increase in cAMP and cGMP [30]. NT also stimulates the hydrolysis of inositol phospholipid in rat brain slices [31]. Similar results were observed in murine neuroblastoma clone N1E115 cells [32-34], in a process that is likely to involve a GTP-binding regulatory protein [32,33] that is sensitive to pertussis toxin. It is possible that our observed alterations in BRR response may simply reflect a hemodynamic response to the changes in systemic arterial pressure induced by the various peptide treatments. This possibility was deemed unlikely, based on several reasons. First, the degree of increase in SAP elicited by phenylephrine at various time intervals after i.c.v, administration of NT, (D-Trp~)-NT, bestatin or aCSF was not compromised (cf. Table I). Second, although both NT and (D-Trp~I)-NT produced a reduction in basal heart rate, these two treatments promoted respectively a suppression and an enhancement of the BRR response. Furthermore, the temporal effect of NT and (D-Trp~)-NT on basal SAP or HR and BRR response did not parallel each other. Third, we previously demonstrated [27] that no significant alterations in the response of the BRR was observed over the range of 8-40 mmHg increase in MSAP, induced by an i.v. infusion of dopamine (15 #g/kg per min). Fourth, Heesch and Carey [35] reported that BRR exhibits acute resetting in both nomotensive and hypertensive rats.

35 That our study was carried out under pentobarbital anesthesia, which is known to depress the BRR, may be another confounding factor. While we do not have experimental results to directly assess this factor, the statistic treatment of our data appeared to minimize its contribution. We compared the temporal action ofpeptide(s) against the vehicle, with the assumption that both groups of animals were under similar influence by pentobarbital during the course of the experiment. It should also be mentioned that rats anesthetized with pentobarbital [15] are actually more sensitive to the vasodepressor effect of i.c.v, delivered NT than conscious animals. Moreover, centrally administered NT inhibits pentobarbital metabolism only in mice but not in rats [36]. That NT is involved in synaptic processes within the central nervous system is quite f'Lrrnlyestablished. NT immunoreactivity is present in cell bodies, fibers and terminals in many regions of the central nervous system [ 10-12,14]. Radioimmunoassay [37,38] for the tridecapeptide also revealed a heterogeneous distribution in the brain and pituitary. Brain NT is concentrated in synaptosomal subcellular fractions [39], is released upon depolarization of the presynaptic membrane [40] and its binding to synaptic membranes from rat brain exhibits high specificity, saturability and reversibility [41]. Autoradiographically [13] identified presumed NT binding sites are in general correspondence with the immunocytochemical distribution of NT projections. As the terminal site for baroreceptor afferents [ 10,25], the NTS is a logical site upon which neurons that contain NT may elicit their modulating effect on the BRR. The synaptic machinery for such an action already exists, with the presence of NT terminals and binding sites at this nucleus [ 10,13]. The feasibility of this notion was confirmed in the present study with the demonstration of a respective inhibitory and enhancing effect of NT and its antagonist on the BRR response, upon microinjection into the bilateral NTS. Preliminary data from our laboratory suggest that NT may reduce the responsiveness of baroreceptive neurons in the NTS to perturbations of arterial pressure. This mode of action, as well as the origin of the NT-containing neurons and the underlying process of signal transduction, however, await further delineation.

Acknowledgements This research was supported in part by research grants NSC-78-0412-B010-04 and NSC-78-0412-B010-46 from the National Science Council, Taiwan, Republic of China, to S.H.H.C.

References 1 Carraway,R. and Leeman,S. E., The isolationof a new hypotensivepeptide, neurotensin,from bovine hypothalami,J. Biol. Chem., 248 (1973) 6854-6861. 2 Bissette, G., Manberg, P., Nemeroff, C.B. and Prange, A.J., Jr., Neurotensin, a biologicallyactive peptide, Life Sci., 23 (1978) 2173-2182. 3 Clineschmidt, B.V., McGuffin,J.C. and Bunting, P.B., Neurotensin antinociresponsive action in rodents, Eur. J. Pharmacol.,54 (1979) 129-139. 4 Kalivas, P.W., Nemeroff, C.B. and Prange, A.J., Jr., Neuroanatomical site-specific modulation of spontaneous motor activityby neurotensin,Eur. J. Pharmacol.,78 (1982) 471-474.

36 5 Martin, G.E., Naruse, T. and Papp, N.L., Antinociceptive and hypothermic actions of neurotansin administered centrally in the rat, Peptides, 1 (1981) 447-454. 6 Nemeroff, C.B., Luttinger, D. and Prange, A.J., Jr., Neurotensin: central nervous system effects of a neuropeptide, Trends Neurosci., 3 (1980) 212-214. 7 Quirion, R., Rioux, F., Regoli, D. and St-Pierre, S., Neurotensin-induced coronary vessel constriction in perfused rat hearts, Eur. J. Pharmacol., 55 (1979) 221-223. 8 Rioux, F., Kerouac, R., Quirion, R. and St-Pierre, S., Mechanisms of the cardiovascular effects of neurotensin. In C.B. Nemeroff and A.J. Prange, Jr. (Eds.), Neurotensin, a Brain and Gastrointestinal Peptide, Ann. N.Y. Acad. Sci., Vol. 400, New York, 1982, pp. 56-74. 9 Rosell, S., Burcher, E., Chang, D. and Folkers, K., Cardiovascular and metabolic actions ofneurotensin and (Gln4)-neurotensin, Acta Physiol. Scand., 98 (1976) 484-491. 10 Higgins, G.A., Hoffman, G.E., Wray, S. and Schwaber, J.S., Distribution of neurotensin-immunoreactivity within baroreceptive portions of the nucleus of the tractus solitarius and the dorsal vagal nucleus of the rat, J. Comp. Neurol., 226 (1984) 155-164. 11 Hokfelt, T., Everitt, B.J., Theodorsson-Norheim, E. and Goldstein, M., Occurrence of neurotensinlike immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, J. Comp. Neurol., 222 (1984) 543-559. 12 Jennes, L., Stumpf, W.E. and Kalivas, P.W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. Comp. Neurol., 210 (1982) 211-224. 13 Kessler, J. P., Moyse, E., Kitabgi, P., Vincent, J.-P. and Beaudet, A., Distribution ofneurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study, Neuroscience, 23 (1987) 189-198. 14 Uhl, G.R., Goodman, R.R. and Snyder, S.H., Neurotensin-eontaining cell bodies, fibers and nerve terminals in the brain stem of the rat: immunohistochemical mapping, Brain Res., 167 (1979) 77-91. 15 Rioux, F., Quirion, R., St-Pierre, S., Regoli, D., Jolicoeur, F.B., Belanger, F. and Barbeau, A., The hypotensive effect of centrally administered neurotensin in rats, Eur. J. Pharmacol., 69 (1981) 241-247. 16 lwata, T., Hashimoto, H., Hiwada, K. and Kokubu, T., Changes of plasma renin activity by intracerebroventricular administration of biological active peptides in conscious rats, Clin. Exp. Hypertens. (A), 6 (1984) 1055-1066. 17 Shido, O. and Nagasaka, T., Effects ofintraventricular neurotension on blood pressure and heat balance in rats, Jap. J. Physiol., 35 (1985) 311-320. 18 Sumners, C., Phillips, M. I. and Richards, E.M., Central pressor action of neurotensin in conscious rats, Hypertension, 4 (1982) 888-893. 19 Lin, K. S., Chan, J.Y.H. and Chan, S. H. H., Reduction in baroreceptor reflex response by angiotensin III and its modification by IleT-angiotensin III and bestatin in the rat, Neurosci. Lett., 90 (1988) 172-176. 20 Checler, F., Amar, S., Kitabgi, P. and Vincent, J.-P., Catabolism of neurotensin by neural (neuroblastoma clone N 1E 115) and extraneural (HT29) cell lines, Peptides, 7 (1986) 1071-1077. 21 Rich, D.H., Moon, B.J. and Harbeson, S., Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes, J. Med. Chem., 27 (1984) 417-422. 22 Umezawa, H., Aoyagi, T., Suda, H., Hamada, M. and Takeuehi, T., Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes, J. Antibiot. (Tokyo), 29 (1976) 97-99. 23 Quirion, R., Rioux, F., Regoli, D. and St-Pierre, S., Selective blockade of neurotensin-induced coronary vessel constriction in perfused rat hearts by a neurotensin analogue, Eur. J. Pharmacol., 61 (1980) 309-312. 24 Rioux, F., Quirion, R., Regoli, D., Leblanc, M.-A. and St-Pierre, S., Pharmacological characterization of neurotensin receptors in the rat isolated portal vein using analogues and fragments of neurotensin, Eur. J. Pharmacol., 66 (1980) 273-279. 25 Miura, M. and Reis, D. J., Termination and secondary projections of carotid sinus nerve in the cat brain stem, Am. J. Physiol., 217 (1969) 142-153. 26 Hutchinson, J. S. and Chan, J. Y. H., Inhibition of central angiotensin II induced drinking by GABA in the rat, Clin. Exp. Hypertens. (A), 6 (1984) 1777-1780. 27 Chan, J.Y.H., Chan, S.H.H., Chen, C.F. and Barnes, C.D., Effects of hestatin on the central cardiovascular regulatory mechanisms in the rat, Regul. Pept., 18 (1987) 75-84.

37 28 Smythe, H. S., Sleight, P. and Pickering, G. W., Reflex regulation of arterial pressure during sleep in man: a quantitative method of assessing baroreflex sensitivity, Circ. Res., 24 (1969) 109-211. 29 McDonald, J.K., An overview of protease specificity and catalytic mechanisms: aspects related to nomenclature and classification, Histochem. J., 17 (1985) 773-785. 30 Mistry, A. and Vijayan, E., Differential responses of hypothalamic cAMP and cGMP to substance P and neurotensin in ovariectomized rats, Brain Res. Bull., 18 (1987) 169-173. 31 Goedert, M., Pinnock, R.D., Dowries, C.P., Mantyh, P.W. and Emson, P.C., Neurotensin stimulates inositol phospholipid hydrolysis in rat brain slices, Brain Res., 323 (1984) 193-197. 32 Amar, S., Kitabgi, P. and Vincent, J.-P., Stimulation of inositol phosphate production by neurotensin in neuroblastoma N 1E115 cells: implication of GTP-binding proteins and relationship with the cyclic GMP response, J. Neurochem., 49 (1987) 999-1006. 33 Bozou, J.-C., Amar, S., Vincent, J.-P. and Kitabgi, P., Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma NIE115 cells: involvement of the inhibitory GTP-binding component of adenylate cyclase, J. Pharmacol. Exp. Ther., 29 (1986) 489-496. 34 Snider, R.M., Forray, C., Pfenning, M. and Richelson, E., Neurotensin stimulates inositol phospholipid metabolism and calcium mobilization in routine neuroblastoma clone N1E-I15, J. Neurochem., 47 (1986) 1214-1218. 35 Heesch, C.M. and Carey, L.A., Acute resetting of arterial baroreflexes in hypertensive rats, Am. J. Physiol., 253 (1987) H974-H979. 36 Widerlov, E., Bissette, G. and Nemeroff, C. B., Centrally administered neurotensininhibits pentobarbital metabolism in mice but not in rats, Neurosci. Lett., 77 (1987) 311-315. 37 Carraway, R. and Leeman, S.E., Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem., 251 (1976) 7045-7052. 38 Kobayashi, R.M., Brown, M. and Vale, W., Regional distribution of neurotensin and somatostatin in rat brain, Brain Res., 126 (1977) 584-588. 39 Ahmad, S., Kwan, C. Y., Vincent, J.-P., Grover, A. K., Jung, C.Y. and Daniel, E.E., Target size analysis of neurotensin receptors, Peptides, 8 (1987) 195-197. 40 Iversen, L.L., Iversen, S.P., Bloom, F., Douglas, C., Brown, M. and Vale, W, Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature, 273 (1978) 161-163. 41 Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., Morgat, J.L., Menez, A., Leeman, S. and Freychet, P., Neurotensin: specific binding to synaptic membranes from rat brain, Proc. Natl. Acad. Sci. USA, 74 (1977) 1846-1850.