Decreased ANF atrial content and vascular reactivity to ANF in spontaneous and renal hypertensive rats

Life Sciences, Vol. 41, pp. 341-348 Printed in the U.S.A.

Pergamon Journal

DECREASED ANF ATRIAL CONTENT AND VASCULAR REACTIVITY TO ANF IN SPONTANEOUS AND RENAL HYPERTENSIVE RATS H. de Le6n, G. Castafieda-Hern~ndez and E. Hong Secci6n de Terap~utica Experimental Depto. de Farmacologla y Toxicologla Centro de Investigaci6n y de Estudios Avanzados, IPN Apartado Postal 22026 14000 Mfixico, D.F. (Received in final form May 13, 1987) Summary The relationship between ANF activity and hypertension was determined by measuring ANF atrial content and vascular reactivity in two different models: spontaneous hypertensive rats (SHR) and renal hypertensive rats (RHR). Atrial extracts and aortic strips were prepared from hypertensive and normotensive animals. Relaxant activities of extracts, synthetic ANF and nitroglycerin were assayed on superfused aortic strips previously contracted by norepinephrine. ANF atrial content was statistically significantly lower in both models of hypertension, presumably by increased ANF release into the circulation which results in depletion of tissue storage sites. Vascular subsensitivity to ANF and nitroglycerin was found in both models of hypertension. Diminished ANF vascular reactivity in hypertension could be due to receptor down-regulation and/or to a decrease in the ability of cGMP to induce relaxation. In 1981 de Bold and colleagues reported that injection of atrial extracts produced potent natriuresis and diuresis (i). The active material, referred as Atrial Natriuretic Factor (ANF), is of protein nature and it is produced by atrial myoendocrine cells (2). The observation that ANF also exhibits a potent relaxant activity on several vascular smooth muscles precontracted with different agonists (3) has been confirmed with synthetic material (4). This action is endothelium-independent (5, 6) and it is not mediated by or ~ adrenergic receptors nor by prostaglandins (7). ANF increases tisular cGMP levels (6), hence it has been suggested that its mechanism of action is similar to that of nitrates (6, 8). In addition to its natriuretic and vasorelaxant activities, ANF is also a potent inhibitor of aldosterone secretion (9). In view of its properties, there has been a growing interest on the role of ANF on blood pressure regulation and on its possible implications in hypertension. However, the information available on these issues is not clear. Sonnenberg et al (i0) using natriuresis as bioassay, and Gutkowska et al (II) using radioimmunoassay (RIA), observed a reduction in ANF content in spontaneous hypertensive rats (SHR), although opposite results were reported by Winquist et al (8), using precontracted aortic rings as bioassay. Vascular reactivity to ANF has been described to be diminished in SHR (8) and renal hypertensive rats (RHR) (12), but ANF injection produced higher natriuretic and hypotensive responses on SHR than on 0024-3205/87 $3,00 + .~0 Copyright (c) 1987 Pergamon Journals Ltd.

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Wistar-Kyoto (WKY) rats (13). To gain more information about the relationship between hypertension and ANF activity, the relaxant effects of atrial extracts prepared from SHR, RHR and from their normotensive controls, of synthetic ANF and of nitroglycerin were assayed on aortic strips from SHR, RHR and normotensive controls contracted by norepinephrine. The results show that both, atrial content and vascular reactivity to ANF are decreased in the two models of hypertension. Materials and Methods Animals. Renal (one kidney) hypertensive female Wistar rats were prepared by constriction of one kidney (through a cotton thread ligature placed in an "8" shape) and contralateral nephrectomy as described by Grollman (14). Non-operated normotensive Wistar rats served as controls. Animals had free access to food and water and were 12 weeks old at the time of the experiments (body weight 200-300 g). SHR from either sex and their respective controls, normotensive WKY were 16-22 weeks old at the time of the experiments (body weight 200-355 g). Systolic blood pressure measured by the tail cuff procedure was 160-230 mm Hg for hypertensive animals and 110-130 mm Hg in the case of normotensive rats. Preparation of extracts. Atrial and ventricular extracts were prepared from different experimental groups. Hearts were obtained from freshly killed rats, atria and ventricular apices from the same hearts were dissected and washed in chilled NaCI 0.9% solution, dried by three successive passages on filter paper and weighed. Extraction was carried out by a modification of the method described by Weselcouch et al (15). Briefly, tissue was homogenized in 10 vol of i M acetic acid and centrifuged at 10,500 g for 30 min. The pellet was rehomogenized, centrifuged as above and pooled supernatants were passed through a SEP-PAK C18 cartridge previously equilibrated with 5 ml of methanol and 5 ml of 1 M acetic acid. The cartridge was washed with 20 ml of 1 M acetic acid and the active fraction was eluted with 2 ml of a mixture of methanol: 1 M acetic acid 80:20 v/v, solvent was evaporated under a gentle nitrogen stream and the dry residue stored at -20°C. This procedure was used since it has been reported that extraction with 1 M acetic acid avoids artifactual proteolysis (16, 17). Extract was dissolved in 0.9% NaCI solution. Dilutions were made immediately before the assays. Doses are expressed as the extract amount equivalent to 1 g (wet weight) of original tissue (g tissue/dose). Atrial extracts produced a potent natriuretic and diuretic effects when injected to anesthetized rats and relaxed precontracted vascular smooth muscle preparations, while ventricular extracts were devoided of these activities. Incubation with trypsine resulted in the complete inactivation of atrial extracts. Bioassay. Relaxant activities of atrial extracts, synthetic ANF and nitroglycerin were assayed on superfused aortic strips. Rat thoracic aortae were dissected and cut in helical strips. One end of each strip was tied to an isometric force transducer (Grass FT 03) and the other to the bottom of a superfusion tissue chamber. Chambers possessed an external jacket in which hot water was circulated to keep tissues warm. Strips were subjected to an initial tension of 2 g and superfused with Krebs solution at 37°C at a flow rate of 5 ml/min. When more than one strip was used, chambers were arranged in a cascade fashion (18). Strips were allowed to stabilize for two hours; during this time, tension was repeatedly adjusted to 2 g. Then, they were contracted by an infusion of norepinephrine (10-SM); once

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the contractile plateau was achieved, increasing doses agents dissolved in 0.5 ml were applied at 5 min intervals.

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Determination of ANF atrial content. Five batches of atrial extracts were prepared from each of the following animals: SHR, WKY rats, RHR and Wistar rats. Each batch was obtained from 11-15 animals. The relaxant activity of each batch was assayed on superfused aortic strips from WKY rats. Determination of vascular reactivity. To determine vascular reactivity to native ANF, two atrial extract batches, one from 20 WKY rats and other from 25 SHR, were assayed on aortic strips from WKY and SHR; then, an atrial extract batch from 20 normotensive Wistar rats was assayed on aortic strips from normotensive Wistar and RHR. In another group of experiments, the relaxant effects of synthetic ANF and of nitroglycerin were assayed on WKY and SHR strips. Additionally, nitroglycerin was also assayed on normotensive Wistar and RHR strips. In all experiments, strips from normotensive rats and their normotensive controls were placed in a cascade fashion, allowing simultaneous determination of vascular reactivity. A total of 6 experiments were performed for each experimental series, the order of the strips was alternated in each experiment to eliminate bias. Reagents. Krebs solution composition was (in mM): NaCI 118, NaHC03 25, KCI 4.7, MgSO 4 1.2, KH2PO 4 1.2, CaCI 2 2.5, Na2EDTA 0.03 and glucose 11.7. The solution was bubbled by a mixture containing 95% 02, 5% CO 2. Norepinephrine hydrochloride was obtained from Sigma (Saint Louis Mo.) and nitroglycerin from Lilly (Indianapolis, In.). Synthetic ANF L-364,343 of 26 aminoacids corresponding to rat ANF (8-33) was a gift from Dr. H.J. G6mez, Merck, Sharp & Dohme Research Labs. (Rahway, N.J.). Statistics. Data are presented as mean values ± SEM. EDs0's and 95% confidence limits (c.l.) were determined from dose-response curves and compared by the student's "t" test as described by Tallarida and Murray (19). Results ANF atrial content. Relaxant effects of atrial extract batches prepared from the four groups of animals were assayed on superfused norepinephrine contracted WKY aortic strips. Dose-response curves obtained with WKY and SHR atrial extracts and those obtained with the normotensive Wistar and RHR atrial extracts are shown in Fig. i. Dose-response curves constructed with the extracts from hypertensive animals were shifted to the right in comparison with those from normotensive controls, EDs0 (95% c.l.) values were 1.3 (0.7-2.4) for WKY, 5.0 (3.7-6.7) for SHR, 1.3 (0.8-2.0) for normotensive Wistar and 4.4 (4.1-4.7) mg tissue/dose for RHR atrial extracts. Differences between WKY and SHR and between Wistar and RHR were statistically significant (p < 0.05). However, no statistically significant difference was observed between the two groups of normotensive nor between the two groups of hypertensive rats. These data indicate reduced ANF atrial content in the two models of experimental hypertension studied. Vascular reactivity. The relaxant effect of an atrial extract batch from WKY rats was assayed on superfused aortic strips from WKY and SHR placed in a cascade arrangement and contracted with norepinephrine. WKY strips were more sensitive than SHR strips (Fig. 2, left). EDs0 (95% c.l.) values were 0.9 (0.8-1.0) and 2.0 (1.4-2.9) mg tissue/dose respectively, being

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Fig. 1 Relaxant effects of atrial extracts made from SHR (left), RHR (right) and their correspondent normotensive controls on aortic strips obtained from WKY rats contracted with norepinephrine. Each dose-response curve was constructed with five atrial extract batches.

statistically significantly different (p < 0.05). Similar results were observed when a SHR extract batch was assayed. ED50 values were 3.7 (2.4-5.9) mg tissue/dose on WKY strips and 7.4 (6.5-8.4) mg tissue/dose on SHR strips, being also statistically significantly different (p < 0.05). As expected from previously mentioned results, the WKY extract was more potent than the SHR one. An atrial extract batch prepared from normotensive Wistar rats was assayed on aortic strips from normotensive Wistar and RHR placed in a cascade. Strips from normotensive animals resulted more sensitive than those of RHR (Fig. 2, right). EDs0 values were I.I (0.7-1.7) and 5.3 (4.4-6.5) mg tissue/dose, respectively. The difference was statistically significant (p < 0.05). The relaxant activity of synthetic ANF was assayed on WKY and SHR aortic strips arranged in a cascade. As observed with the atrial extracts, strips from WKY were more sensitive than those from SHR (Fig. 3, left) EDs0 values were 0.87 (0.65-1.17) and 3.72 (2.09-6.61) x 10 -7 M respectively, being statistically significantly different (p < 0.05).

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WKY EXTRACT

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Fig. 2 Relaxant effects of an atrial extract from WKY rats on superfused aortic strips from WKY and SHR (left) and of an atrial extract from normotensive Wistar rats on aortic strips from normotensive Wistar and RHR (right). Aortic strips were precontracted with norepinephrine. Symbols correspond to mean values ± SEM determined on 6 strips.

Aortic strips from normotensive rats were also more sensitive to the relaxant effect of nitroglycerin (Fig. 3, right). On superfused strips from WKY and SHR placed simultaneously in a cascade, EDs0 values were 0.96 (0.85-1.07) and 2.95 (2.89-3.01) x 10 -6 M respectively. When the cascade was formed with superfused strips from normotensive Wistar and RHR, EDs0 values for the nitrate were 0.34 (0.29-0.40) and 1.22 (i.05-1.41) x lO -6 M respectively. Differences between EDs0 values on strips from hypertensive rats and normotensive controls were statistically significant

(p < 0.05). Discussion The atrial content of ANF was assessed by the capacity of atrial extracts to relax aortic strips from WKY rats previously contracted with norepinephrine. The amounts of ANF found in atria from both types of hypertensive animals, spontaneous and renal, were statistically significantly lower than those found in atria from correspondent normotensive controls. These data are in agreement with the reduced atrial content of ANF observed in SHR, when the peptide was evaluated by means of its natriuretic effect (I0) or by radioimmunoassay (ll).

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SYNTHETIC

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Fig. 3 Relaxant effects of synthetic ANF (left) and nitroglycerin (right) on superfused aortic strips from WKY and SHR precontracted with norepinephrine. Symbols correspond to mean values ± SEM determined on 6 strips.

It is interesting to point out that a decrement in atrial content of ANF was found in both, spontaneous and renal hypertensive rats, a characteristic shared by DOCA-salt hypertensive rats (20). Therefore, the possibility that hypertension could be due to a decrease in the ANF atrial content does not seem likely, since the mechanisms involved in the genesis and maintenance of the three types of hypertension is probably different. On the other hand, the postulation that ANF release is a compensatory mechanism induced by hypertension, ending in a depletion of the peptide atrial content (I0), is compatible with the experimental findings described. Furthermore, the higher ANF plasma levels found in SHR (II) and DOCA-salt hypertensive rats (20) provides additional support to the above contention. Vascular reactivity to ANF appears to be lower in hypertensive than in normotensive rats, as indicated by the subsensitivity of aortic strips from SHR and RHR to the relaxant effect of both, native and synthetic ANF. Schiffrin and coworkers have reported that the number of ANF binding sites is decreased in several models of hypertension (12, 21). They have suggested that the subsensitivity to ANF might be due, at least in part, to a downregulation phenomenon induced by increased ANF plasma levels (12).

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Vascular reactivity to nitroglycerin was also found to be diminished in SHR and RHR. It is well known that the vasodilator profiles of ANF and nitrates are similar, relaxation being mediated by an increase of tissue cGMP levels (6, 8). However, ANF binds to Specific binding sites and activates the particulate form of guanylate cyclase, whereas nitrates have a direct action mainly in the soluble form of the enzyme (22). Thus, although it cannot be ruled out that subsensitivity to ANF and to nitrates could be produced by different pathways, it seems likely that decreased vascular reactivity to ANF in hypertension is also mediated by postreceptor events, such as the ability of cGMP to induce intracellular calcium efflux (23). Therefore, decrease in ANF atrial content as well as vascular subsensitivity to ANF found in both models of experimental hypertension, SHR and RHR, seem to be a consequence of the increased release to ANF into the circulation. This appears to be a compensatory response to hypertension of insufficient effectiveness. Acknowledgements. This study was supported by grant 24/37 from COSNET of the Secretary of Public Education. H. de Le6n is a CONACYT fellow. Autors thank Mr. J. S~nchez and J.J. L6pez for the technical assistance and Mrs. G. Loeza for secretarial assistance. References I. A.J. DE BOLD, H.B. BORENSTEIN, A.T. VERESS and H. SONNENBERG. Life Sci. 28:89-94 (1981). 2. A.J. DE BOLD. Proc. Soc. Exp. Biol. Med. 170:133-138 (1982). 3. M.G. CURRIE, D.M. GELLER, B.R. COLE, J.G. BOYLAN, W.Y. SHENG, S.W. HOLMBERG and P. NEEDLE~N. Science 221:71-73 (1983). 4. R.J. WINQUIST, E.P. FAISON and R.F. NUTT. Eur. J. Pharmacol. 102: 169173 (1984). 5. R. SCIVOLETTO and M.H.C. CARVALHO. Eur. J. Pharmacol. i01: 143-145 (1984). 6. R.J. WINQUIST, E.P. FAISON, S.A. WALDMAN, K. SCHWARTZ, F. MURAD and R.M. RAPOPORT. Proc. Natl. Acad. Sci. USA 81:7661-7664 (1984) 7. R. GARCIA, G. THIBAULT, M. CANTIN and J. GENEST. Am. J. Physiol. 247: R34-R39 (1984). 8. R.J. WINQUIST, E.P. FAISON, E.P. BASKIN, P.B. BUNTING, R.F. NUTT and L.T. CALLAHAN III. J. Hypertension ~ (Suppl. 3): 325-327 (1984). 9. K. ATARASHI, P.J. MULROW, R. FRANCO-SAENZ, R. SNAJDAR and J. RAPP. Science 224:992-994 (1984). 10. H. SONNENBERG, S. MILOJEVIC, C.K. CHONG and A.T. VERESS. Hypertension ~: 672-675 (1983). Ii. J. GUTKOWSKA, K. HORKY, C. LACHANCE, K. RACZ, R. GARCIA, G. THIBAULT, O. KUCHEL, J. GENEST and M. CANTIN. Hypertension 8 (Suppl. I): 1-1371-140 (1986). 12. E.L. SCHIFFRIN, J. ST-LOUIS, R. GARCIA, G. THIBAULT, M. CANTIN and J. GENEST. Hypertension 8 (Suppl. I): 1-141-1-145 (1986). 13. S.C. PANG, M. HOANG, J. TREMBLAY, M. CANTIN, R. GARCIA, J. GENEST and P. HAMET. Clin. Sci. 69:721-726 (1985). 14. A. GROLLMAN. Proc. Soc. Exp. Biol. Med. 57:102-104 (1944). 15. E.O. WESELCOUCH, W.R. HUMPHREY and J.W. AIKEN. Am. J. Physiol. 249: R595-R602 (1985). 16. A.J. DE BOLD and T.G FLYNN. Life Sci. 33:297-302 (1983).

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17. N.C. TRIPPODO, R.D. GHAI, A.A. MACPHEE and F.E. COLE. Biochem. Biophys. Res. Commun. 119:282-288 (1984). 18. E. HONG. Prostaglandins 8:213-220 (1974). 19. R.J. TALLARIDA and R.B. MURRAY. Manual of Pharmacologic Calculations with Compute r Programs. p. 9-19, Springer-Verlag, New York, (1981). 20. M. KIHARA, M. KIHARA and E.L. BRAVO. (abstract), Fed. Proc. 45: 755

(1986). 21. E.L. SCHIFFRIN and J. ST-LOUIS. Clin. Invest. Med. 8:A127 (1985). 22. R.J. WINQUIST. Fed. Proc. 45:2371-2375 (1986). 23. L.M. POPESCU, C. PANOIU, M. HINESCU and O. NUTU. Eur. J. Pharmacol. 107:393-394 (1985).