Brain Research, 586 (1992) 289-294

289

Elsevier Science Publishers B.V.

BRES 17934

The role of angiotensin, and A T 2 receptors in the presser, drinking and vasopressin responses to central angiotensin D a v i d C. H o g a r t y , E l i s a b e t h A. S p e a k m a n , Viviana Puig a n d M. Ian Phillips Department of Physiology, Collegeof Medicine, Unirersity of Florida, Gainesrille, FL 32610 (USA) (Accepted 3 March 1992)

Key words: Angiotensin receptor; Losartan; Vasopressin: Presser; Drinking

Angiotensin I! tAng ii) given centrally produces an increase in blood pressure and motivation to drink. The physiological mechanisms that mediate the presser response include release of vasopressin (AVP) and activation of the sympathetic nervous system. Using 2 new Ang II receptor antagonists, we were able to investigate the role of AT t or AT., receptors in mediating these effects. Adult male Sprague-Dawley rats were cannulated in the lateral ventricle and 5 days later catheterized in the carotid artery for blood pressure measurements. All experiments were carried out in conscious rats. Three treatments were given intraventricularly (i.v.t.), in 2 pl artificial cerebrospinal fluid (ACSF) at 30 rain intervals: (1) 50 ng Ang il, (2) 0.7/zg AT I antagonist Losartan or 7.0 p.g AT2 antagonist PD123177, followed by 50 ng Ang ll, and (3) 50 ng Ang ll, to test for recovery. Blood pressure and drinking measurements were recorded. Also, blood samples for assay of AVP were drawn at I or 3 rain post-injection in 2 separate groups of rats. We found that both Losartan and PDI23177 significantly reduced release of AVP to Ang I I i rain post-injection. Losartan significantly blocked the presser response (P < 0.001), while PD123177 had no significant effect. Drinking was also antagonized by Losartan (P

< < @}

< .~ o.

10

PD123177-treated animals Pressor response. Fig. 4 shows the effect of pretreatANG II

i__

__

DUP753

PD 123177

TREATMENT Fig. 2. Release of AVP measured ! rain after treatment with Ang Ii alone (50 ng) or with Ang ii followed by either Losartan (0.7/~g) or PDI23177 (7.0/.¢g). Treatment with Ang !I alone (n = 6) increased AVP to 24.2+4.0 pg/ml. The levels of AVP were significantly reduced ( P < 0,05) to 14.4-t-3.3 pg/ml when Losartan (n = 7) was

given before Ang !I (P < 0.05), After pretreatment with PD 123177 (n = 6) plasma AVP levels were significantly reduced (P < 0.05) to 16,2+4,0 pg/ml,

pg/ml. Administration of Losartan (n - 8) before Ang II tended to reduce AVP release (2.0 + 0.5 pg/ml, n.s,).

Dipsogenic response, F~g. 3 shows the effect of Losartan on the drinking response to Ang II (n = 7). Ang ll (50 /~g) caused an immediate dipsogenesis with an intake of 6,(} :t: 1,1 ml in a 30 rain period. Following pretreatment with Losartan, drinking in response to Ang II was

ment with PD123177 on the pressor response to central Ang II ii~jection (n = 5). Ang II alone caused a 13.9 + 1.3 mmHg rise in MAP. When PD123177 was given prior to Ang II, there was no significant attenuation of the pressor response (11.5 5:3.3 mmHg). The pressor response to the second Ang II injection was 17.6 + 1.6 mmHg. Plasma AVP. As with the Losartan treated animals, samples were drawn from 2 different groups of rats at different time points after treatment. The data for 1 rain post-injection are shown in Fig. 2. When Ang II was given alone, plasma A V P levels were 24.2 +4.0 p g / m l (same as above). After pretreatment with PD 123177 (n = 7) plasma A V P levels were significantly reduced ( P < 0.05) to 16.2 + 4.0 p g / m l . Samples were also collected 3 min after pretreatment with PD123177 (n = 6), Injection of Ang I1 (i.v.t.)

2O I% F~e~s ,ooo~

,¢se¢

T --

r~e~u'¢l¢

I,

~

T

E

,p~s~e~t ,,,,

~0

"'" pl,l,l,l, ,I,I,,,I° ;I;I;IIl~

0 ANG II

•

DIJP'753

.

ANG II

.

TREATMENT Fig. 3. The effect of Losartan (0.7 #g) on the drinking response to Ang II (50 ng) was meast~red in 7 animals. Ang II caused an immediate dipsogenesis (6.0+ 1.1 ml in 30 min). Following pretreatment with Losartan, water intake was significantlyreduced to 2.7 + 0.9 ml (P < 0.05). Thirty min later, drinking to Ang II was not significantly different to that after the initial dose.

ANG II

pD123177

ANG II

"

TREATMENT

Fig. 4. The effect of PDI23177 (7,0/~g) on the pressor response to Ang II (50 ng)was measured in 5 rats. Treatment with Ang !! caused a 13.9-t-1.3 mmHg increase in MAP. The increase in MAP measured after pretreatment with PD123177 (11.5 + 3.3 mmHg)was not significantly different from the response to Ang I! alone. The pressor response to a second Ang !i injection given 30 min later was 17.6+ 1.6 mmHg, which was not significantly different from the first dose.

292 10.

prior injection of PD123177 (2.9 + 1.4 ml) compared to the response to Ang It alone (5.9 + 2.3 ml). The dipsogenie response to the second Ang It injection (without PD123177) was not significantly different from the first (2.8 _+0.9 ml), though the reductions in water intake in both cases were similar (2.7 _+0.9 ml after Losartan, vs. 2.9 _+ 1.4 ml after PD123177). It should also be noted that the large variance in the control group (Fig. 5) could account for the lack of statistical significance.

g'

8' 7 6¸ 5-

z

4-

ne

3" 2'

DISCUSSION

1 ANG II

ANG II

PD123177 TREATMENT

Fig. 5. The effect of PD123177 (7.0 ~g) on the drinking response to Ang !1 was measured in 5 rats. The mean water intake was reduced (but not significantly) by pretreatment with PDI23177 (2.9± 1.4 mi) compared to the response to Ang !! alone (5.9 ± 2.3 ml). The dipsogenie response to the second Ang I! injection (without PDI23177) was not significantly different from the first (2.8 ± 0.9 ml).

released 3.3 + 0.4 pg/ml (same baseline data as above). Pretreatment with PD123177 resulted in an AVP plasma level of 3.8 + 0.6 pg/ml, which does not differ significantly from AVP levels after Ang !! (i.v.t.) alone. All AVP values measured 3 min post-injection were significantly reduced ( P < 0 . 0 5 ) compared to those measured I rain post-injection, Dipsogenic response. Fig, 5 shows the effect of PD123177 on the drinking response to Ang !! (tt - 5), The water intake to Ang I! was reduced (but not significantly) by

16

TACHYPNYLAXI$ OF" DRINKING RESPONSE TO ANG II IVT

14 12 E O Z

10

----------

B'

::::::::::::::::::::: ' ......... '

6,

' ....................

i:i:~:~:i:i:i:i:i:!:i ..........

z

~.

T

:.'.,.,.:qq.;.:.;.;

' iii!i!i!!i!iil!il~

0

1

, 2

3

INJECTION (50 ng Ang II Ivt at 30 rain, intervals) Fig, 6, The drinking response to 3 successive doses of Ang !! (50 ng) was measured to test for tachyphylaxis in 3 rats, The mean water intake to the fiat dose was 10,4 ± 2,8 ml, to the second 4,2 ± 1,0 ml, and to the third 1,2 ± 0,8 ml,

The results show that blockade of central Ang II receptors inhibits the pressor response to Ang II and that this inhibition is mainly due to specific antagonism of the ATm receptor subtype. Since the central p~essor response has an AVP component we tested the release of AVP by central administration of Ang I!. The AVP response to Ang I1 (i.v.t.) is high in the first minute after injection, then lessens by 3 min following treatment. Blockade of either AT~ or AT 2 receptors significantly attenuated AVP release to Ang I1 (i.v.t.), measured 1 rain post-injection. We also found a decrease in drinking to Ang !! after pretreatment with Losartan and recovery 30 min later. PDI23177 also decreased mean water intake, but the decrease was not significant. The effect of Losartan on the pressor response to Ang il (i,v,t,) was also investigated in a study by Wong et el, :t, They treated rats with 3.5 Izg Losartan (i,v,t,) prior to injection of 0.35 ~g Ang il, and round a significant attenuation of the pressor response, Our results confirm this finding, but the difference being that we chose lower doses of antagonist (0.7 ~g Losartan) and agonist (50 ng) to approximate more closely the physiological actions of Ang !!. Because of the small volume of CSF in the brain, the concentration of ~g injections of drugs reaches supraphysiological levels. Further studies by our group have shown that AT~ receptors mediate the central Ang 11 stimulated AVP release and pressor responses in the spontaneously hypertensive rat (SHR) tT. Since the aim of the study was to analyze AVP release to central Ang I1 and the mediation by receptors of the central pressor response to Ang ll, we have not attempted to address the question of peripheral effects on the brain in this study. Central injection of Ang II causes the release of AVP 8 and diuresis 6 more effectively than peripheral Ang II injection 6 Therefore, the intraventricular Ang II injections probably acted on central Ang II receptors. The main target sources of AVP release by Ang II

293 are the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus. The specificity of Ang II induced release of AVP from the hypothalamus has been demonstrated. Sladek and Joynt ~6 showed that AVP release by Ang II in the rat hypothalamoneurohypophyseal system in organ culture was inhibited by the angiotensin antagonist Sar~Alas Ang II. Electrophysiological studies have shown that Ang I1 acts specifically on the SON to release AVP 9.12. In a study using hypothalamic slices, Losartan (the AT t antagonist) inhibited the depolarizing action of Ang II on SON neurons ~2. The effect of Ang II on the SON neurons is direct and not mediated by synaptic input because the SON cells were depolarized by Ang I! in Ca 2÷ free medium which would eliminate synaptic transmission ~2. The present results extend this finding of inhibition of the Ang II electrophysiological response by an AT~ antagonist in the SON, by showing that AT~ blockade partially inhibits AVP release. There is no information on the effects of AT,` blockade on the SON or the intracellular actions of Ang !I on the PVN. Taken together, the intracellular effects of AT~ blockade on SON cells and the attenuation of extracellular release of AVP by AT~ antagonism provide strong evidence for Ang I! acting on AT~ receptors in the SON. However, there also appears to be a role of the AT,` receptors in the control of AVP release because PD 123177 was also effective in reducing the AVP release to Ang 11. As no functions for AT, receptors have been found previously, this result may represent the first report of a functional role for AT 2 receptors. The location of the AT,` receptors involved in the AVP release is far from clear. The regional distribu. tion of Ang II receptor subtype binding sites in the brain have been shown by autoradiography using Losartan and PD123177 ins.J9. Studies with suifhydryl reducing agent dithiothreitol, which selectively inhibits AT I receptors, have corroborated these findings 3,1s,20. AT I receptors predominate in areas of the brain which mediate cardiovascular responses to Ang II (i.v.t.) These include the SON and PVN of the hypothalamus, the circumventricular organs (subfornical organ, area postrema, OVLT, median eminence) and the NTS m~s. AT,` receptors have a different distribution and have not been associated with these so-called cardiovascular sites in the brain ~s. Since neither AT l nor AT 2 antag. onism alone completely inhibited AVP release, it is possible that both ATI and AT 2 receptors are involved synergistically. The release of AVP in response to central Ang II is prompt. We collected blood samples for AVP assay at 2 different times (1 and 3 min) after injection. We

found that plasma AVP levels at 3 min after treatment were significantly (P < 0.05) decreased when compared to levels measured at 1 rain post-dose. Keil et al. ~ measured AVP in trunk blood collected 90 s after 50 ng Ang II (i.v.t.). Using radioimmunoassay (RIA) they determined plasma AVP to be 22.6 _+5.6 pg/ml. This is comparable to the present experiment, where we measured levels of 24.2 _+4.0 pg/ml 1 rain following the same dose of Ang II (i.v.t.). However, we found that at 3 rain the amount of AVP released by 50 ng Ang II was 3.3 :l: 0.40 pg/ml. Bealer et ai. ~ measured AVP by RIA in trunk blood collected 2 min after 500 ng Ang II (i.v.t.). The level measured was 10 IzU/mi, which is equivalent to 19.2 pg/ml, for a dose 10 times higher than used in this study. Thus there appears to be a rapid decline or clearance in total AVP released 60-90 s after Ang II injection. In addition to AVP release, other mechanisms play an important role in mediating the presser response to central Ang II. Central administration of Ang II increases sympathetic nerve activity, which could be responsible for the early phase of the blood-pressure response 5. In this study, Losartan completely blocked the presser response to Ang II, while only moderately reducing AVP release. We also found that PD123177 reduces Ang II release of AVP, but has no significant effect on the Ang II presser response. It has been shown that the vasopressor action of AVP is masked by an intact baroreflex 4, so the full effect of AVP would have to be studied in relation to the baroreflex. Another consideration is that the sympathetic activation may be fully blocked by Losartan, while unaffected by PD123177. It will be interesting to measure plasma catecholamines before and after Ang 11 receptor blockade so that the relative importance of sympathetic output to the presser response can be ascertained. Blockade of either receptor subtype individually, failed to completely suppress the dipsogenesis produced by Ang II (i.v.t.). As the protocol required administration of the Ang II antagonist after the initial dose of Ang II, one concern was tachyphylaxis with repeated doses ~a. We tested for tachyphylaxis in a group of 3 rats. The data from that experiment suggest a possible tachyphylaxis of the drinking response after repeated doses of Ang II. This complicates the interpretation of data that show a significant decrease in drinking after pretreatment with Losartan. Our results take into account tachyphylaxis and show inhibition of Ang II stimulated dipsogenesis by Losartan but not significantly by PD123177, suggesting that both receptor subtypes may be involved in mediating tile dipsogenic response to Ang If. Higher doses were not tested because they would not be relevant to understanding

~4 the physiological action of Ang II. At higher concentrations the receptor antagonists cannot be assured to act only on Ang !1 receptors. In summary, using the new nonpeptide antagonists for Ang 11 receptor subtypes we have found that the central pressor response to Ang II (i.v.t.) is completely blocked by Losartan, the AT~ receptor antagonist. The release of AVP by Ang II (at 1 min post-dose) was significantly reduced by blockade of either the AT I or the AT., receptor subtype. This result is the first report of a functional effect of AT., receptors. Drinking to Ang ll produced tachyphylaxis but there was still a significant attenuation of central Ang ll induced drinking by Losartan, though the decrease with PD123177 was not significant. We concluded that the central pressor response to Ang ll may be mediated by the AT a receptor subtype. The AVP component of the pressor response may be mediated by both ATt and AT, receptors and the drinking in response to Ang il (i.v.t.) appears to be mediated primarily by ATe, but because of rapid tachyphylaxis the mechanism for this response needs to be further defined in future experiments. ,4ck,,wh.d/,,ements. This ruscarch was supported by NIH Grant IROIIIL27334. W~ would like to acknowledge the technical assistante of Birgitta Kimura and Jomlthan Bui We would also like to thank Dr. Ron Smith at DuPont for the gifts of Losartan, Dr. J. Keiser at Warner Lambert for the PDI23177, and Gayle Butt~rs for word processing,

REFERENCES I Beal~r, S.L., Phillips, M,I., Johnson, A.K. and Schmld, P.G,, Anleroventral third ventricle lesions reduce antidiuretic responses to an~iotensin I!, Am, J, PhysioL, 236 (1979) E('H0=E(HS, 2 Bumpus, F.M,, Catt, K.J., Chiu, A,T,, deGasparo, M,, Goodfriend, T., Husain, A., Peach, MJ., Taylor, D,G. and Timmer. roans, P.B.M.W.M., Nomenclature for angiotensin receptors: a report of the nomenclature committee of the council for high blood pressure research, tlypertcv~siot¢, 17 ( 1991) 720-721, 3 Chiu, A,T., Herblin, W,F., McCall, D.E,, Ardecky, R,J,, Carini, DJ., Duncia, J.V., Pease, L.J,, Wong, P,C,, Wexler, R,R., Johns,m, A.L. and Timmermans, P.B.M,W.M,, Identification of angiotensin 11 receptor subtypes, Biochem. Biophy,~', Res. ('omm,~l,, 165 (1989) 196-2()3, 4 Cowley, A.W., Monos, E. and Guyton, A,C,, Interaction of vaso-

pressin and the baroreceptor reflex system in the regulation of arterial blood pressure in the dog, Circ. Res., 34 (1974) 505-514, 5 Falcon, J.E., Phillips, M.I., Hoffman, W.E. and Brody, M.J., Effects of intraventricular angiotensin I! mediated by the sympathetic nervous system, Am. J Physiol., 235 (1978) H392. 6 Hoffman, W.E., Phillips, M.I., Schmidt, P.G., Falcon, J. and Weet, J.R., ADH release and the pressure response to central angiotensin It and cholinergic stimulation, Neuropharmacology, 16 (1977) 463-472. 7 Jhamandas, J.R., Lind, R.W. and Renaud, L.P., Angiotensin may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat, Brain Res., 487 (1989) 52-61. 8 Keil, L.C., Summy-Long, J. and Severs, W.B., Release of vasopressin by angiotensin !!, Emiocrinology, 96 (1975) 1063-1065. 9 Nicoll, R.A. and Barker, J.L., Excitation of supraoptic neurosecretory cells by angiotensin !1, Nature New Biol,, 233 (1971) 172-173. 10 Phillips. M.I., Functions of brain angiotensin, Atom. Re~'. of PhysioL. 49 (1987) 413-435. II Phillips. M.I., Brain Angiotensin. in P, Gross (Ed,), Circumrentricular Organs and Body Fhdds, Vol. i!i, CRC Press, Boca Raton, FL, 1987, 163-182. 12 Phillips. M.i., Yang, C.R., Bourque, C.W. and Renaud, L.P., Angiotensin !! depolarizes supraoptic neurones recorded in rat hypothalamic explants, Soc. NeuroscL Abstr., 16 (1990) Abstract 286.4. 13 Quirk, W.S., Wright, J,W. and Harding, J.W., Tachyphylaxis of dipsogenic activity to intracercbmveatricular administration of angiotensins, Brai, Res,, 452 (1988) 73-78. 14 Raft, H., Kane, C.W. and Wood, C,E,, Arginine vasopressin responses to hypoxia and hypercapnia in late gestation fetal sheep, Am, J PhysioL, 260 (1991) RI077 -RI081. 15 Rowe, B.P., Grove, K.L., Saylor, D,L, and Speth, R.C., Discrimination of angiotensin II subtypes in the rat brain using nonpeptid~ receptor antagonists, Regal, Pept., 452 (1991) 73-78. 16 Shldek, C,D. and Joynt, RJ,, Angiotensin stimulation of vasoprcssin reluL~se from the rat hypothalamoneurohypophyseal system in organ culture, I",doc'ri,olop, y, 104 (1979)148, 17 Sp~ttkmLtn, E.A,, tlogarty, D,, PUil~,V, and Phillips, M,i., Central anBtolensin I! type ! r~ceptor blockade with Losartan in the splmtaneously hypertc;nsiv¢ rat ($HR), I"ASE'B ,I,, f~ (1992) 541h, 18 Spcth, R,C,, Rowe, B,P,, Grove, K,L,, Carter, M,R, and Saylor, D,, Sulfllydryl reducing agents distinguish two subtypes of angiotensin Ii receptors in the rat brain, Brai, Rt,s,, 549 (1991) 1=8, 19 Tsutsumi, T, and Saavedra, J,M,, Quantitative autoradiography reveals different angiotensin I! receptor subtypes in selected rat brain nuclei, J, New,rex'he,m,, 56 (1991) 348-351, 20 Whitebread, S,, Mele, M,, Kamber, B, and deGasparo, M., Preliminary biochemical characterization of two angiotensin Ii receptor subtypes, Biorhem, Biophys, R¢,,~, Ccmmmn,, 163 (1989) 284-291, 21 Wong, P,C,, Price, A,W,, Chiu, A,T,, Duncia, J,V,, Carini, D.J., Wexler, R,R,, Johnson, A,L, and Timmermans, P,B.M,W.M,, Nonpeptide angiotensin !1 receptor antagonists Viii, Characleri. zation of functional antagonism displayed by DuP 753, an orally active antihypertensive agent, J, Pharmacol, 'Exp. Ther,, 252 (1989) 719-725,