Effect of chronic administration of the converting enzyme inhibitor enalapril (MK 421) on brain atrial natriuretic peptide receptors in Wistar-Kyoto and spontaneously hypertensive rats

134

Brain Research. 475 (1988) 134- 140

Elsevier BRE 23222

Short Communications

Effect of chronic administration of the converting enzyme inhibitor enalapril (MK 421) on brain atrial natriuretic peptide receptors in Wistar-Kyoto and spontaneously hypertensive rats Adil J. Nazarali, Jorge S. Gutkind, Fernando M.A. Correa and Juan M. Saavedra Unit on Preclinical Neuropharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.)

(Accepted 16 August 1988) Key words: Autoradiography;Brain nucleus; Renin angiotensin system; Angiotensin convertingenzyme; Hypertension; Receptor;

Atriopeptin; Atrial natriuretic factor

Spontaneously hypertensive rats (SHR) showed lower brain ANP binding density when comparedwith normotensive Wistar-Kyoto (WKY) rats. In the WKY, angiotensin convertingenzyme inhibitor, enalapril (25 mg/kg, p.o. for 14 days), decreased the number of ANP binding sites selectively in the subfornical organ and area postrema. Conversely,enalapril increased ANP binding density in the SHR, but only in the area postrema. Enalapril has central effects on ANP binding sites, specificto the circumventricularorgans. The atrial natriuretic peptide (ANP) plays a role in fluid metabolism and regulation of blood pressure through peripheral but also central mechanisms 4'2°. Spontaneously hypertensive rats (SHR), a model of human essential hypertension 18 have alterations in ANP metabolism. In SHR, plasma levels of ANP have been demonstrated to be high, possibly as a consequence of increased peptide release from the atria to the circulation 16. Systemic administration of ANP produced a decrease in blood pressure in SHR 5. In addition, there is a marked decrease in ANP binding sites in SHR, both in peripheral tissues 2°'21 and in the brain 20,24. Through its diuretic and natriuretic effects, and its inhibition of vasopressin and aldosterone release, ANP has effects opposite to those of the vasoactive, water conservatory peptide angiotensin II 2'4~20. Physiological and pharmacological evidence indicates that these two peptide systems are interrelated, both in the periphery and in the brain 2°'22. In SHR, the lower number of ANP binding sites observed in the subfornical organ corresponds to a higher number of

angiotensin II receptors 24. We have asked the question whether treatment of SHR with an inhibitor of the angiotensin converting enzyme (ACE), enalapril 6 which decreases blood pressure presumably through inhibition of angiotensin II formation, will result in changes in ANP binding sites in SHR, and whether these changes would be similar or different to those appearing in normotensive WKY rats. We have found that chronic treatment with enalapril resulted in a specific decrease in ANP binding sites in circumventricular organs of WKY, whereas SHR showed a change in the opposite direction, that is an increase in ANP binding sites, localized exclusively to the area postrema. The two strains of rats (male, 12-week-old), normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR), used in this study were obtained from Taconic Farms (Germantown, NY). The animals were housed in standard laboratory metal cages in a temperature-controlled room (24 + 1 °C) with a 12 h on/12 h off lighting schedule. Rat chow and water were provided ad libitum. The two groups

Correspondence: J.M. Saavedra, Laboratory of Clinical Science, National Institute of Mental Health, 9000 Rockville Pike, Bldg. 10, Room 2D/45, Bethesda, MD 20892, U.S.A.

135 (WKY and SHR) of respectively 16 animals each, were randomly separated into a treated (n = 8) and untreated control (n = 8) groups. Whereas the treated group from both strains of animals were administered a daily oral dose of enalapril (25 mg/kg, 1 ml/100 g) for 14 days, the untreated control animals were given an equivalent volume of vehicle (distilled water) per kg weight, for the same period. Mean systolic blood pressures in all groups of rats were measured with an electrosphygmomanometer (Narco Bio-systems, Houston, TX) coupled to photoelectric sensors (IlTC, Landing, N J) and recorded on a Model 7 Grass polygraph. Measurements were taken on day prior to the start of the experiment and on days 3, 6, 9 and 14 of treatment. Animals were killed by decapitation between 09.00 and 11.00 h, 24 h after the last dose on day 14. Blood was immediately collected in ice-cold tubes containing E D T A (for measurement of angiotensin I and plasma renin activity) or a portion collected in ice-cold heparinized tubes (for measurement of ACE activity). The tubes were subsequently centrifuged and plasma separated prior to the biochemical analysis. Brains were rapidly dissected out, meninges removed, and the brain tissue immediately frozen in isopentane on solid carbon dioxide (-30 °C). Tissue samples were stored at -70 °C and within 24 h tissue sections of 16 pm thickness were cut in a cryostat set at -14 °C. The tissue sections were thaw-mounted onto cold gelatin-coated glass microscope slides and stored in a dessicating jar under vacuum at 4 °C for 24 h. For the measurement of plasma ACE activity, a 1 mM concentration of [14C]hippuryl-L-histidyl-L-leucine (3 mCi/mmol, New England Nuclear, Boston, MA) was used as substrate 17. Plasma sample blanks (10pl) were heated at 95 °C for 5 min prior to analysis. All plasma samples were incubated at 37 °C for 15 rain after the addition of 20 pl of 0.1 M Tris-HCl buffer, pH 7.4 and a 20/d of 0.75 M NaC1. Reaction was stopped with the addition of 50pl of 1 N HC1 and the [14C]product extracted from the acidic medium with water-saturated ethyl acetate. Plasma renin activity (PRA) and plasma angiotensin I (ANG I) concentrations were measured by Hazelton Laboratories, Vienna, VA following the method of Menard and Catt 15. For the characterization of ANP binding sites, con-

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Fig. 1. Systolic blood pressure measurements (mm Hg) in control-SHR ( A - - - A ) , enalaprit-treated SHR ( A - - - A ) , control-WKY ((3------0), and enalapril-treated WKY (0------0). Values are means + S.E.M. from 8 animals per group. * Indicates P ~<0.05 in treated SHR vs control SHR. secutive tissue sections were preincubated in a buffer composed of 50 mM Tris-HC1 buffer, pH 7.4, containing 100 mM NaCI, 5 mM MgC12, 0.5% bovine serum albumin, 40 pg/ml bacitracin, 4 pg/ml leupeptin, 2 pg/ml chymostatin, and 0.5 pg/ml phenylmethylsulfonylfluoride. Tissue sections were subsequently incubated for 60 rain at room temperature in fresh buffer containing a saturating concentration (0.3 nM) of 3-[125I]iodotyrosy128 rat ANP (spec. act. 2050 Ci/ mmol, Amersham, Arlington Heights, IL) s. Adjacent sections were incubated with 1 p M unlabelled ANP (rat atrial peptide, 28 amino acids, Peninsula Laboratories, Belmont, CA) to determine the nonspecific binding. After incubation, tissue sections were washed in ice-cold 50 mM Tris-HC1 buffer, 3 times for 2 rain each, and subsequently in ice-cold distilled water for 1 min. All tissue sections were quickly dried under a stream of cold air. After incubation, the dry tissue slides were placed in X-ray cassettes (CGR Medical Corp., Baltimore, MD) and apposed against single coated LKB 3H-UItrofilms (LKB Products, Rockville, MD). Depending on the optical density of the ANP binding sites in the sections examined, film exposure time usually ranged from 4 to 7 days (at room temperature). The films were subsequently developed with a D19 Kodak developer for 4 min at 4 °C. Quantitation of ANP binding sites could be performed by including a set of 16 pm thick brain paste standards containing known quantities of [125I]ligand with each sheet of film. Standards were prepared as described earlier 9. Sections from the same brain regions of the control

136 and treated rats were exposed on the same sheet of film. The optical densities of the autoradiograms were d e t e r m i n e d by computerized microdensitometry and subsequently converted to fmol [125I]ANP bound/mg protein of standard 9 after correcting for 125I decay. Values were expressed as X + S.E.M. D a t a were subjected to a two-way analysis of variance according to the e x p e r i m e n t a l design followed by post hoc analysis with N e w m a n - K e u i s test. Values of P < 0.05 were considered significant; however, in the case of receptor alterations a Bonferroni adjustment was p e r f o r m e d and P < 0.007 were considered significant. A d u l t S H R h a d much higher b l o o d pressures than their age-matched normotensive W K Y controls (Fig. 1). Plasma A C E was lower in untreated S H R when c o m p a r e d to the u n t r e a t e d W K Y rats. Plasma A C E activities in the S H R and W K Y animals were 240 + 12 and 372 + 36 nmol/mg protein/h, respectively (P ~< 0.05). In contrast, untreated S H R did not show significant differences in either A N G I or P R A when c o m p a r e d with untreated W K Y rats. The plasma A N G I concentrations in the W K Y and S H R rats were 0.8 _+ 0.1 and 1.1 _+ 0.1 ng/ml, respectively. P R A was 4.2 _+ 0.4 ng/ml/h and 4.0 + 0.4 ng/ml/h in untreated W K Y and S H R , respectively. In all areas studied, u n t r e a t e d S H R had a much

lower A N P binding site concentration than that of untreated normotensive W K Y rats; most areas in S H R showed a 7 0 - 8 0 % lower A N P binding density (Table I). In the internal plexiform layer of the olfactory bulb, the decrease in A N P binding sites in S H R with respect to W K Y was about 40%. In the nucleus of the solitary tract of the SHR, A N P receptors could not be detected (Table I). Treatment with enalapril decreased blood pressure in S H R from day 6 onwards, and until the end of the treatment period at day 14. No difference in blood pressure were noted in W K Y during the enalapril treatment (Fig. 1). Treatment significantly inhibited plasma A C E activity by nearly 85% to 42 _+ 12 and 48 + 12 nmol/mg protein/h in S H R and W K Y animals, respectively. Enalapril treatment elevated plasma A N G l levels to 1.8 + 0.5 and 2.0 + 0.5 ng/ml in the W K Y and S H R , respectively (P ~< 0.05). P R A increased significantly after treatment in both W K Y (10.0 _+ 2.4 ng/ml/h) and S H R (8.2 + 2.0 ng/ml/h) (P ~< 0.0l vs untreated rats of each strain). Enalapril treatment had differential effects in brain A N P binding sites in W K Y and SHR. The changes observed were in all cases restricted to the circumventricular organs. Brain A N P binding density was lower in the subfornical organ and the area postrema of the treated W K Y animals c o m p a r e d to untreated W K Y animals (Fig. 2, Table 1). Converse-

TABLE I Apparent ANP binding density in discrete rat brain nuclei of control and enalapril-treated Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

All data are presented as mean -+ S.E.M. of 8 animals. F-values for significant difference (P ~< 0.007) from the two-way ANOVA were: subfornical organ, drug = 7.19, strain = 122.26; choroid plexus, strain = 346.8; paraventricular nucleus, strain = 87.57; area postrema: strain = 71.245, drug × strain = 15.63; internal plexiform layer of the olfactory bulb, strain = 91.21; internal granular layer of the olfactory bulb, strain = 106.97. Brain nuclei

Subfornical organ Choroid plexus Paraventricular nucleus Area postrema Nucleus of the solitary tract Internal plexiform layer of the olfactory bulb Internal granular layer of the olfactory bulb *P ~<0.007 compared to control WKY group. **P <~0.007 compared to control SHR group. n.d. = not detected.

Apparent binding density (fmol/mg protein) WKY Control

SHR Control

WKY Treated

SHR Treated

135 +_ 10 152 + 8 41 -+ 4 97 + 6 33 + 3 86 + 2 28 -+ 3

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31 + 2 34 + 3 15_+3 48 -+ 8** n.d. 43-+5 4_+2

137 ly, A N P binding density was increased only in the area postrema of the treated S H R animals (Fig. 3). In S H R and W K Y rats treatment did not alter A N P binding in the choroid plexus, the paraventricular nucleus, the nucleus of the solitary tract, the internal

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plexiform layer or internal granular layer of the olfactory bulb. Our results confirm and expand previous reports 2°'22 of large differences in A N P binding site concentrations in the brain of W K Y and SHR. The present experiments, including drug treatment were performed twice, and in each case similar results were obtained. Genetically hypertensive rats had

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Fig. 2. Autoradiographic images of [125I]ANPbinding in the rat brain of control WKY at the levels of: (A), subfornical organ (SFO), choroid plexus (ChP); (B), paraventricular nucleus (PVN); (C) area postrema (AP), nucleus of the solitary tract (NTS); and (D) internal plexiform layer (IPl), and internal granular layer (IGr) of the olfactory bulb.

Fig. 3. Autoradiographic images of [nSI]ANP binding in the rat brain at the levels of; (A) area postrema (AP) in control SHR and (B) area postrema (AP), in enalaprii-treated SHR; (C), non-specific binding.

138 very low ANP binding densities. In most areas, the number of ANP binding sites in SHR is not more than 20-30c/( than that of normotensive WKY. In addition to decreased ANP binding in circumventricular organs and choroid plexus >2a we report here decreased binding site concentrations in areas inside the blood-brain barrier, such as the paraventricular nucleus, the nucleus of the solitary tract and the olfactory bulb. Thus, lower number of ANP binding sites occurs not only in brain areas accessible to peripherally circulating peptide, but in areas related to the central ANP system as well 2°::. The paraventricular nucleus plays an important role in the formation of the antidiuretic hormone vasopressin eT. The subfornical organ may be involved in the regulation of vasopressin formation and release, through alterations in its receptors to circulating ANP and angiotensin and its projections to the paraventricular nucleus 7'13. ANP decreases vasopressin release into the general circulation e'2s. A lower number of ANP binding sites in these areas may be reflected in a decreased inhibition of vasopressin release. This may be compounded by the increased number of angiotensin II receptors in the subfornical organ of SHR e<2a, since angiotensin II stimulates the release of vasopressin 19. Indeed, vasopressin levels are higher in plasma of SHR, a factor which can contribute to hypertension 3. Both the area postrema and the nucleus of the solitary tract tp- have been implicated in central cardiovascular regulation. The lower number of ANP binding sites in the area postrema of SHR, and their apparent absence in the nucleus of the solitary tract, may reflect a decreased central response to the actions of an antihypertensive peptide. It is of interest that in these areas, the angiotensin II receptor concentrations are higher than in normotensive WKY rats 2°'e2, again pointing to opposite changes in binding sites for these two antithetical peptides in the brain. The alterations of ANP binding sites in the choroid plexus of SHR reported here and previously 2° may be related to changes in cerebrospinal fluid formation, since the peptide has been shown to regulate the function of the choroid plexus epithelium 26. The significance of the reduced number of ANP binding sites in the olfactory bulb of SHR has not yet been clarified.

The antihypertensive effects of enalapril in SHR are well documented 2s. We used a dose previously shown to reduce established hypertension in adult SHR 2s and obtained similar results. Blood pressure was not significantly modified in WKY and the enalapril effects on blood pressure were specific for the hypertensive animals. However, the biochemical changes produced by enalapril in plasma (reduction of ACE activity, increased ANG 1 levels and PRA activity) were similar for SHR and WKY. These resuits confirm earlier studies >'3u'33 and demonstrate that under the conditions of our experiment the formation of peripheral angiotensin II is inhibited, PRA is increased due to the lack of inhibitory action of angiotensin I1-~t,32. It is of interest to note that, untreated SHR had much lower plasma ACE activity than untreated WKY, confirming previous reports ~7, but that the treatment with enalapril reduced enzyme activity in each group to approximately the same levels. Thus, there was apparently no correlation between the ACE plasma levels in treated SHR and WKY and the enalapril antihypertensive effects. In normotensive rats, treatment with enalapril produces a decrease in the number of ANP binding sites which is selective for the circumventricular organs, the subfornical organ and the area postrema. In SHR, changes in ANP binding sites occur also in a cirvumventricular organ, the area postrema, but they are not present in the subfornical organ. The direction of the change, however, is opposite to that in WKY, since SHR show increased number of ANP binding sites. Since these effects are restricted to the circumventricular organs, structures located outside the blood-brain barrier, they may be related to alterations in the peripheral, rather than the central, peptide systems. The mechanisms and the implications of these changes are not fully understood. Plasma ANP levels differ in WKY and SHR after ACE inhibitor treatment. SHR have higher pretreatment plasma ANP levels than WKY 14. After chronic ACE inhibition, ANP levels in SHR significantly decrease, whereas those in the normotensive animals remain unchanged from pretreatment levels m. It is tempting to hypothesize that in normotensive rats, blockade of angiotensin II synthesis produces, by a compensatory mechanism, a decrease in the concentration of ANP binding sites, to maintain fluid balance and blood pressure within normal levels. Indeed. blood pres-

139 sure is not modified in W K Y after enalapril. In genetically hypertensive rats, however, inhibition of angio'tensin II synthesis produces, p r o b a b l y by a compensatory mechanism, a decrease in circulating A N P levels 1°. As a consequence, A N P binding sites in the area p o s t r e m a increase in number. If the area postrema, as it has been postulated 1A2, plays an important role in the regulation of b l o o d pressure in the rat, the increase in A N P binding sites could be important as an additional mechanism tending to reduce hypertension. A similar mechanism of up-regulation of A N P binding sites in cirvumventricular organs has recently been d e m o n s t r a t e d in d e h y d r a t e d rats, which show decreased p l a s m a A N P levels tl and increased circumventricular organ A N P binding density 2°. It is not known, however, why the A N P binding sites in the subfornical organ of S H R do not change in response to changes in plasma A N P in this model. It is possible that part of the A N P binding site alterations in S H R are of genetic origin, since decreased A N P binding has been observed in young, prehyper-

tensive S H R 2°. It is also possible that systemic enalapril t r e a t m e n t would affect angiotensin II receptors and/or formation in the cirvumventricular organs, structures rich in both angiotensin II binding sites 24 and A C E 23.

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In conclusion, our results indicate that chronic systemic t r e a t m e n t with an A C E inhibitor differentially altered, in S H R and W K Y , brain A N P binding site density in circumventricular organs involved in the regulation of fluid homeostasis and blood pressure. These results further suggest the existence of a close interaction between the central and peripheral angiotensin II and A N P systems, and the possibility of a role for cirvumcentricular organ A N P receptors in the regulation of blood pressure.

The authors are grateful to Dr. William L. Henckler of Merck Sharp and D o h m e , Rahway, NJ for the gift of enalapril maleate. A . J . N . is a fellow of the Alberta H e r i t a g e F o u n d a t i o n for Medical Research.

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