Effects of bestatin on the central cardiovascular regulatory mechanisms in the rat

Regulatory Peptides, 18 (1987) 75-84

75

Elsevier RPT 00592

Effects of bestatin on the central cardiovascular regulatory mechanisms in the rat Julie Y.H. Chan 1'2, Samuel H . H . Chan 1'2, C.F. Chen 2 and Charles D. Barnes I 1Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-6520, U.S.A. and 2Department and Institute of Pharmacology, National Yang-Ming Medical College, Taipei 11221, Taiwan.

(Received 18 November 1986;revisedversion received 18 February 1987;accepted26 February 1987)

Summary We evaluated the effects of bestatin, the specific aminopeptidase-B and leucine aminopeptidase inhibitor, on the central cardiovascular regulatory mechanisms in Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular injection of bestatin (100 or 200 nmol/5/~1) consistently elevated the basal systemic arterial pressure and heart rate. At the same time, this degradative enzyme blocker increased the sensitivity of the baroreceptor reflex responses as well as the efficacy of the modulatory actions of the medullary nucleus reticularis gigantocellularis on these reflexes. We speculate that enhancing the tonic activities of the endogenous neuropeptides in the brain by protecting them from their catabolic enzymes may affect the central cardiovascular regulatory machinery by modifying the operations of the baroreceptor feedback controls and their modulatory mechanisms. Bestatin; Degradative enzyme inhibitor; Endogenous neuropeptide; Baroreceptor reflex sensitivity and modulation; Nucleus reticularis gigantocellularis; Rat

Introduction Bestatin ([(2s,3g)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine) is a specific aminopeptidase-B [1-4] and leucine aminopeptidase inhibitor [2-4] isolated from Correspondence: S.H.H. Chan, Institute of Pharmacology,National Yang-Ming MedicalCollege,Taipei

11221, Taiwan. 0167-0115/87/$03.50 © 1987ElsevierSciencePublishers B.V. (BiomedicalDivision)

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Streptomyces olivoreticuli and characterized by Umezawa [3]. Although it has been studied extensively in immunology as a small molecular immunomodifier [3], its biochemical property as a peptidase inhibitor has only begun to be exploited in pharmacologic studies. Apart from its purported involvement in antagonizing the catabolism of the endogenous opioid peptides [5-9] and hence the analgesic process, the functional properties of bestatin have not been extensively addressed. Wright et al. [10] recently reported that intracerebroventricularly administered bestatin potentiates and prolongs the heightened pressor response of spontaneously hypertensive rats to angiotensin III. The present study further examined the thus implied effects of this degradative enzyme blocker on the central cardiovascular regulatory mechanisms. We recognize that the central neural control machinery on circulation may be operated [11-12] either directly via the basic cardiovascular reflexes or indirectly through modulation of these fundamental feedback loops by other neural substrates. Accordingly, the influence of bestatin on both modes of operation was assessed, taking advantage of an experimental paradigm [12] that we previously designed for such an evaluation. We report in this communication that this aminopeptidase inhibitor elevated the basal systemic arterial pressure and heart rate. It also increased the sensitivity of the baroreceptor reflex responses, as well as the efficacy of the modulatory actions on these reflexes of the nucleus reticularis gigantocellularis (NRGC), a medullary site we have determined [12-17] to be intimately related to the physiologic and pharmacologic aspects of cardiovascular regulations.

Materials and Methods

Adult male Sprague-Dawley rats (214-296 g) were used in this study. Under pentobarbital sodium (40 mg/kg, i.p., with 10 mg/kg/h i.v. supplements) anesthesia, intubation of the trachea and cannulation of the right femoral artery and vein were routinely performed to facilitate ventilation, measurement of systemic arterial pressure and administration of drugs. The animal was thereafter securely fixed in a stereotaxic headholder (Kopf), with the body resting on a heating pad. The body temperature was maintained at 37°C through a thermostatically controlled heating system. Following the procedures used in a previous study [18], a 25-gauge stainless-steel cannula was implanted into the lateral cerebral ventricle on the right side at (mm): P 1.0-1.5, R 1.0 1.5 and H 4.0-5.0 with reference to the bregma. I.c.v. injection of bestatin or artificial cerebrospinal fluid (CSF) was carried out via a 25-#1 Hamilton microsyringe connected to the cannula by a PE-20 tubing. A total volume of 5 #1 was delivered over at least 1 min to allow full diffusion of the, solution. The experimental paradigm we employed to evaluate the central neural cardiovascular regulatory actions of the NRGC in a previous study [12] was used. 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 routinely displayed on a Grass polygraph). A single locus in the NRGC (5.0-7.0 mm caudal

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to the lambda, 0.5-1.0 mm lateral to the midline and 7.0-8.5 mm below the cerebellar surface) capable of lowering the H R by 30-50 b/rain upon electrical activation (10s train of 1 ms, 20-50-#A rectangular pulses) was identified at the beginning of each experiment. Reflex bradycardia, used as an index for baroreceptor reflex (BRR) responses, was induced by administering phenylephrine (5 #g/kg) via the cannulated femoral vein. Modulations of the BRRs by the N R G C were assessed by evaluating their interactions when this reticular site was electrically stimulated simultaneously with the induction of the reflex. The effects of bestatin (100 or 200 nmol, i.c.v.) on the central cardiovascular regulatory mechanisms were investigated in two separate series of experiments. In the first series, the action and time-course of the degradative enzyme blocker on basal SAP and H R were studied. The second series examined the effects of bestatin on (a) NRGC-elicited cardioinhibition, (b) BRR response and (c) interactions between (a) and (b). Each set of these 3 tests was executed at 10-rain intervals over 65 rain after administration of the drug into the lateral ventricle. The order of each of these 3 manipulations was altered randomly to avoid sequential dependency. I.c.v. injection of artificial CSF served as the vehicle control in both series. 301 /

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Fig. l. Time course alterations in basal MSAP and H R following i.c.v, administration of bestatin at 100 (11) and 200 (&) nmol, and artificial CSF (O). The initial MSAP and H R of each group were respectively 118.5 + 5.9 and 366.7 + 12.9, 123.7 + 3.8 and 375.0 ± 8.5 and 120.5 4- 4,5, and 357.5 ± 11.2 (mm Hg and b/rain). Values presented are mean ± S.E.M. 01 = 6). Significant difference (P < 0.001) exists between the 3 groups in the ANOVA analysis, and *P < 0.05 and **P < 0.01 vs artificial CSF at comparable time points in the Student-Newman-Keuls' test.

78 Bestatin and phenylephrine solutions were freshly prepared respectively with artificial CSF and saline immediately before use. Verifications of the N R G C stimulating loci and the i.c.v, injection sites were carried out on frozen 30-#m sections stained with Cresyl violet. The sensitivity of BRR responses measured at each time-interval was expressed as a quotient that represents the unit change in H R per unit change in MSAP (b/min per mm Hg). Individual values were further normalized to a percentage of control to compensate for variations between animals. The effects of both doses of bestatin on basal MSAP and HR, NRGC-elicited cardioinhibition, BRRs and N R G C - m o d ulated BRRs were statistically assessed vs. the vehicle control, using the analysis of variance (ANOVA) followed by the Student-Newman-Keuls' test for a posteriori multiple comparisons at corresponding time intervals.

Results

Effects of bestatin on basal cardiovascular responses The animals used in this study had an overall basal MSAP of 121.0 + 5.8 mm Hg and a H R of 370.0 4- 15.7 b/min (mean ± S.E.M., n = 36). The individual values for each group of rats are indicated in the captions to Figs. 1 and 3. While i.c.v, injection of artificial CSF elicited minimal alterations in MSAP and HR, bestatin (100 or 200 nmol/5 #1) promoted hypertensive (F2,1o7 = 59.97, P < 0.001) and tachycardic (F2,1o7 = 15.07, P < 0.001) responses (Fig. 1) that were significantly different (P < 0.05 or 0.01, n = 6) from the vehicle control. Although both curves maximized at 30 min following treatment, the one produced by the higher dose of bestatin had an appreciably larger magnitude as well as a steeper rising and a slower falling slope.

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Fig. 3. Time-course changes in the sensitivity of BRR responses following i.c.v, administration of bestatin at 100 (hatched bar) and 200 (solid bar) nmol, and artificial CSF (open bar). The initial MSAP and H R of each group were respectively 116.4 + 5.5 and 365.3 4- 8.3., 119.7 4- 6.3 and 375.6 4- 3.1, and 125.5 4- 1.9 and 380.0 4- 5.4 (mm Hg and b/min). Values presented are mean + S.E.M. (n = 6). Significant difference (P < 0.001) exists between the 3 groups in the A N O V A analysis, and *P < 0.05 and **P < 0.01 vs artificial CSF at comparable time points in the Student-Newman-Keuls' test.

Effects of bestatin on NRGC-induced bradycardia, BRR response and NRGC-moduiated BRR response Fig. 2 presents typical polygraph tracings illustrating the effects of i.c.v, bestatin (200 nmol) on the NRGC-induced bradycardia, BRRs and NRGC-modulated BRRs. In agreement with our previous findings [12], electrical activation of the N R G C consistently promoted cardioinhibition, associated mostly with hypotension. Furthermore, this reticular nucleus was capable of interacting with the BRR by potentiating the concomitantly induced reflex bradycardia. Activation of the same NRGC locus elicited no significant modifications in the degree of its induced cardioinhibition, during the period when the bestatin-promoted hypertension and tachycardia reached their maxima (30-35 min postinjection). The reflex bradycardia and its potentiation by the NRGC, on the other hand, were both appreciably augmented. Time-course effects of bestatin on the sensitivity of the BRR response and the NRGC-modulated BRR response The temporal alterations in the sensitivity of the BRR responses (Fig. 3) and the NRGC-modulated BRR responses (Fig. 4) to i.c.v, injection of either artificial CSF, or bestatin (100 or 200 nmol) were further investigated in 3 other groups of animals. In contrast to the vehicle control, the aminopeptidase inhibitor significantly (P < 0.05 or 0.01, n = 6) augmented the sensitivity of the BRR responses (F2,125 = 68.01, P < 0.001) and the efficacy of the NRGC in potentiating the reflex bradycardia (F2.125 = 38.74, P < 0.001L in a time course that paralleled its vasopressor and

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POSTINJECTION TIME Cmin) Fig. 4. Time-course changes in the sensitivity of NRGC-modulated BRR responses following i.c.v, administration of bestatin at 100 (hatched bar) and 200 (solid bar) nmol, and artificial CSF (open bar). The initial MSAP and H R of each group were the same as in Fig. 3. Values presented are mean ± S.E.M. (n = 6). Significant difference (P < 0.001) exists between the 3 groups in the A N O V A analysis, and *P < 0.05 and **P < 0.01 vs artificial CSF at comparable time points in the S':.:~nt-Newman-Keuls' test.

cardioexcitatory actions (cf. Fig. 1). Although both temporal patterns of enhancement were similar and maximized around 30-35 min after treatment, the one elicited by 200 nmol of bestatin was significantly larger than that promoted by the lower dose.

Correlations between hypertension and the enhanced sensitivity of the BRR response and the NRGC-rnodulated BRR response by bestatin Significant correlations existed between hypertension and the enhanced BRR sensitivity promoted by both doses of bestatin (r = 0.8900 and 0.7944, P < 0.001 and 0.01 respectively, df = 10). Similarly, the increase in SAP was significantly correlated with the augmentative effects of the degradative enzyme blocker (100 and 200 nmol) on the NRGC-modulated BRR responses (r = 0.9113 and 0.7143, P < 0.001 and 0.01 respectively, df = 10).

Discussion

We demonstrated in the present investigation that, in addition to eliciting hypertension and tachycardia, i.c.v, application of bestatin increased the sensitivity of BRR responses and the efficacy of the medullary N R G C in its modulatory actions on these reflexes. The two latter effects exhibited a time course that paralleled the changes in basal SAP and HR, and were positively and significantly correlated with the induced

81 vasopression. As such, bestatin may effectively influence both modes of operation [11-12] in central neural regulation of cardiovascular functions. As an aminopeptidase inhibitor, it is reasonable to speculate that bestatin may exert its effects on the central cardiovascular regulatory mechanisms by enhancing the tonic endogenous peptide activities in the brain. This speculation is supported by two biochemical observations. First, the maximal inhibitory action of bestatin is not reached until after approximately 30 min of equilibration with the substrate in vitro [19]. This agrees nicely with our demonstrated maximization of its cardiovascular effects at 30-35 min. Second, the reported [2] inhibitory potency of bestatin on aminopeptidase B (Iso = 160 nM) is compatible with the two doses (100 and 200 nmol) of the degradative enzyme blocker used in the present study. Along with being a specific aminopeptidase-B (arginyl aminopeptidase [1]) and leucine aminopeptidase inhibitor [2-4], bestatin may also be a potent blocker of alanyl and leucyl aminopeptidase [1]. Based on these properties and presently available data on amino acid sequences, it follows that the neuropeptides most likely to be protected by this degradative enzyme inhibitor may at least include, albeit not exclusively, neurotensin, somatostatin and substance P. Other authors also suggested enkephalin [6,9] and angiotensin III [10]. While our present data cannot readily identify these candidates and their possible origins as well as sites and modes of action, it should be pointed out that a role in central cardiovascular regulation is quite established for at least angiotensin, enkephalin and substance P (see [20-27] for review). In addition, binding sites and/or immunoreactivity for all these 5 neuropeptides are present [28-34] in the nucleus tractus solitarius, the termination site for baroreceptor afferents [35]. These data, together with our recent finding that the NRGC projects to the same nucleus [15], suggest that an anatomic substrate for our observed changes in the sensitivity and modulations of the BRRs may already exist. Whether the above candidate neuropeptides may actually participate in central cardiovascular regulation in conjunction with bestatin is currently under investigation in our laboratories. We recently obtained preliminary data suggesting a possible role for angiotensin III. Thus, when bestatin (200 nmol) was administered (i.c.v.) together with the angiotensin III antagonist, IleT-angiotensin III (100 nmol), the ordinarily enhanced sensitivity of the BRR responses (cf. Fig. 3) was further increased. More interestingly, this augmentation exhibited a time course that paralleled the reduction of BRR sensitivity elicited by exogenously applied angiotensin III (100 pmol, i.c.v.). It is conceivable that, with a positive correlation, the enhanced sensitivity of the BRR responses and their modulations by the NRGC may simply be a direct consequence of the heightened SAP. Although we are unable to confirm or refute this possibility, an explanation of our observations based entirely on hemodynamic mechanisms is unlikely. For example, Garey et al. [36] recently reported that the baroreceptors retain their ability to reset acutely in normotensive and experimental hypertensive rats. Furthermore, in control experiments in which the arterial pressure was elevated by 8-40 mm Hg with an i.v. infusion of dopamine (15 #g/kg/min), we did not find a significantly altered sensitivity for BRRs tested over this range. The above view may also account for another seeming discrepancy in the present

82

study, viz. because of the enhanced BRR sensitivity, one might expect that bradycardia instead of tachycardia should accompany the bestatin-induced hypertension. It should be pointed out that the design of this study calls for an acute induction of the BRR against a background of tonic hypertension (cf. Fig. 2). Thus, our observations may reflect the sum influences of various endogenous peptides that may differentially affect the phasic and tonic loop gain of the feedback mechanism. A partial support for this contention exists in the opioid system. For example, the decarboxy analog of Leu-enkephalin (RX783016), a relatively specific/~-receptor agonist, decreases at low dose [37], while the x-receptor agonist, ketocyclazocine, increases [27] the BRR sensitivity. In conclusion, bestatin may affect the central cardiovascular regulatory machinery by modifying the operations of the baroreceptor feedback controls and their modulatory mechanisms. Speculatively, this may be achieved by enhancing the tonic endogenous neuropeptide activities in the brain through protection from their degradative enzymes. Since bestatin is by itself a peptide and an immunomodifier [3], it is also at least intuitively feasible that it may promote its central cardiovascular effects directly via these properties. Many neuropeptides are now known to act on lymphocytes as immune mediators [38], and the ability of bestatin to stimulate the immune system is suggested to be an expression of its ability to bind to, and inhibit, cellsurface aminopeptidases [2]. It thus seems likely that bestatin (or its analogs) might in fact function as a CNS neuromodulator. These various modes of action and the central substrates involved, however, require further elucidation.

Acknowledgements Supported in part by Research Grant NSC76-0412-B010-23 from the National Research Council, Taiwan, to S.H.H.C.

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