376

Brain Research, 126 (1977) 376- 381 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

Evidence for direct neuronal stimulation by intraventricul~r angiotensin II

WILLIAM E. H O F F M A N , PHILLIP G. S C H M I D and M. IAN PHILLIPS

Departments of Physiology and Biophysics, and Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242 (U.S.A.) (Accepted January 20th, 1977)

Angiotensin 1I (All) is a hormone which has diverse actions in the rat. When given intravenously (i.v.) in low doses, AII will produce a pressor response by direct vasoconstriction 5. When infused intraventricularly (IVT) AII will produce antidiuretic hormone (ADH) releasel4,1~, drinkinglg,24, 25 and a centrally mediated pressor response 4,13,24. Since AII is such a potent vasoconstrictor agent it has been suggested that the effects produced by central AII are due to local vasoconstriction in the brainS,19,26. It was the purpose of this experiment to test this hypothesis by evaluating the effects of ventricular infusions of a vasoplegic agent, papaverine, on the central pressor and A D H releasing effects of All. Sprague-Dawley rats weighing 300-400 g were used. They were implanted with a chronic lateral cerebral ventricular cannula, femoral artery and vein catheters and a chronic urinary bladder catheter according to the method of Hoffman et al. 14. On the day of the experiment, an intravenous (i.v.) infusion of a hydrating solution containing 10 m M sodium chloride, 5 m M sodium bicarbonate and 130 m M glucose was started at a rate of 0.20 ml/min. The intravenous infusion was continued for the duration of the experiment. The animals were awake and unrestrained during testing. During the experiments, continuous measures of urine conductance, urine flow, blood pressure and heart rate were recorded. Antidiuresis is defined here as a simultaneous decrease in urine flow and an increase in urine conductance. Antidiuresis occurred consistently with i.v. A D H and IVT AII infusions in the rats undergoing a water diuresis in these experiments. Release of A D H to IVT AII injections was estimated in unanesthetized animals according to the methods of Hoffman et al. 14. Briefly, standard curves were developed in each animal for urine conductance changes to a range of doses of A D H from 0.5 to 4 mU infused i.v. Urine conductance changes to IVT AII for these same animals were then plotted on individual standard curves produced for A D H in order to establish the amount of endogenous A D H released 21. For all animals normalized urine conductance changes were linear when plotted against logl0 A D H dose, r ---= 0.88, slope ~ 0.75, agreeing with data previously reportedl4, 21. Absolute changes in urine conductance ranged from 7 3 ± 8 mho for 0.5 mU to 326~:39 mho for 4 mU. All values are reported as m e a n ~ S . E . Eight animals were tested with 50 ng AII IVT injections alone and immediately

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Fig. I. Cardiovascular and antidiuretic effects of 50 ng All IVT injections in rat No. 1449 before and immediately after a 5 min IVT infusion of papaverine (30 Mg/ml) at a rate of 30 Mg/min. Papaverine pre-infusion was given through the same intracranial cannula as used for All injection. Urine conductance changes corresponded to 2.08 mU ADH equivalent release in first test and 1.95 mU after papaverine. after a 5 min IVT infusion of papaverine in a concentration of 15 mg/ml at a rate of 1 /I/min. Eight animals were tested as above using a 30 mg/ml papaverine infusion given at a rate of 1 /l/rain. Four of these animals were tested first with 15 mg/ml papaverine and are part of the first group. These particular animals were thus tested with 50 ng All alone and after each of the 2 doses of papaverine given 1VT. The IVT infusion o f papaverine in concentrations of 15 mg/ml or 30 mg/ml given at a rate of 1 #l/min for 5 rain had no effect on blood pressure or heart rate (Fig. 1): papaverine 15 mg/ml blood pressure change ~ 1~1 mm Hg, heart rate ~ - - 1 0 @ 7 / m i n " papaverine 30 mg/ml blood pressure change 2 ~ 1, heart rate =- 10±6/min. Likewise, neither o f these two doses of papaverine produced antidiuresis or a change in urine conductance : 15 mg/ml - 13:k 10 mho, 30 mg/ml 8 ± 4 mho, indicating that A D H was not released. Central pre-infusions of" papaverine also had no significant effect on A D H release or the pressor action of IVT All infusions through the same intracranial cannula (Fig. 2). The solution of papaverine which had no effect on central All responses produced a complete blockade of the vascular effects of All in hindlimb perfusion experiments, in 5 rats, paFaverine, in a solute concentration of 0.3 mg/ml, produced a decrease in hindquarters' perfusion pressure from 9 7 ~ 6 mm Hg to 584:11 mm Hg, and abolished all direct vasoconstrictor effects of All. Before papaverine infusion, All (5 Mg) infused into the hindquarters produced an increase in perfusion pressure o f 34:-k3 mm Hg. After papaverine, however, 5 Mg A l l produced an increase in perfusion pressure of only 2 ~ I mm Hg. These data are consistent with the results of Schmid et al. m~ and indicate both the potency and the ability of papaverine in blocking the vasoactivity of All. in order to show that the central antidiuretic and pressor effects of All could be blocked, the specific A l I antagonist, saralasin acetate, Sara-alaS-All (Norwich Pharmacal) was used. Six rats were tested with 500 ng All IVT injections before and after a

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Fig. 2. ADH release and pressor response to 50 ng All IVT infusions before and immediately after a 5 rain IVT infusion of 15 mg/ml papaverine or 30 mg/ml papaverine. There were no significant antidiuretic or cardiovascular effects to pre-infusions of papaverine (see text). 5 min IVT infusion of saralasin at a rate of 1 #g/#l/min. In these animals the antidiuretic and pressor responses to 1VT All injections were completely abolished by central administration of saralasin. In the first test, without saralasin, the pressor response and A D H equivalent release to 500 ng All 1VT were 16:~:1 mm Hg and 1.64-! 0.20 m U respectively, in the second test, immediately after a 5 rain infusion of saralasin, these responses were 2 i I m m Hg and 0.2 E0.12 m U : both Values were significantly decreased from the first test (P ~:+~0.00l). Therefore, All injected into the cerebral ventricles produces a centrally mediated increase in systemic blood pressure and antidiuretic effect which can be blocked by central pre-infusions of specific All antagonist, saralasin. Bickerton and Buckley ~ reported the centrally mediated pressor effects of All in dogs in 1961. Since that time it has been repeatedly suggested that the central effects of All are due to vasoconstriction and not specific receptor stimulation s,19,26,'~7, although this has been disputed by several other authors 9,16,22. The evidence arguing for or against the possibility that central vasoconstriction mediates the central responses has been mostly indirect. Barrett 3 showed that intravascular AH infusion does produce an increase in cerebral vascular perfusion pressure in the cat, suggesting cerebral vasoconstriction. However, in the rat, Mandel and SapirsteinlS found no increases in cerebrovascular resistance during intravenous All infusions.

379 Papaverine produces smooth muscle relaxation which is independent of muscle innervationZ,2,11, it antagonizes all stimuli that constrict smooth muscle al. It has repeatedly been shown to produce cerebral vasodilation 17, and is effective in counteracting cerebral vasospasms when applied locally 7 or intravascularly 1~. In experiments reported here, intravascular infusions of papaverine into the rat hindlimb produced vasodilatation and completely blocked the vasoconstrictor effects of All. This is confirmatory evidence of the vascular effects of papaverine. However, IVT infusions of papaverine had no cardiovascular or antidiuretic effects by themselves and did not attenuate the central All induced responses. Since both papaverine (molecular weight 375) and All (molecular weight = 1044) are water soluble, their distribution within the ventricular space should have been similar. Thus, IVT injections of AII would be expected to reach the same cerebrovascular smooth muscle elements as papaverine, particularly since the central All responses are usually initiated within 10 sec li. Finally, the lack of effect of central papaverine infusions on A D H release and pressor effects produced by All contrast with the inhibitory effect of the vasoplegic agent on All induced drinking. In the same dose range as used here Nicolaidis and Fitzsimons 19 reported that papaverine inhibited drinking initiated by All. This may indicate a different mechanism by which central All mediates drinking as opposed to blood pres:,~ure and A D H release effects. Under identical infusion parameters to that of IVT papaverine, ventricular saralasin application completely blocked the antidiuretic and pressor effects of angiotensin. This indicates that All is acting centrally to initiate these responses and that the pre-infusion procedure with saralasin adequately blocked central All receptors. That a specific All antagonist was able to abolish central All responses while the potent cerebral vasodilator and vasoplegic agent, papaverine 17, had no effect is an indication that angiotensin acts on specific periventricular receptors and that its actions are not secondary to local cerebral vasoconstriction. These results are in agreement with the recent report of the existence of angiotensin !I containing nerve terminals in the brain of the rat by Fuxe et al. ~°. A high density of terminals containing All was found in areas known to be of importance in central cardiovascular control, such as the sympathetic lateral column of the spinal cord and the locus coeruleus in the midbrain, in addition, a moderate density ofaxosomatic contacts was reported in the dorsomedial nucleus and ventral segment of the hypothalamus, a region identit'..'ed as containing the periventricular All receptors for thirst and blood pressure effects in the rat 6,~a,20. In summary, it has been shown in these experiments that papaverine, a demonstrated vasodilatory and vasoplegic agent, has no cardiovascular or antidiuretic effects when infused into the cerebral ventricles of the rat. Central administration of papaverine also has no inhibitory effect on subsequent All induced pressor and antidiuretic responses. Central periventricular All receptors can be blocked by pretreatment with a specific All antagonist with infusion parameters similar to those used for central papaverine tests. We conclude that the blood pressure and antidiuretic responses to IVT All are mediated by neural connections to these receptors and are not due to local vasoconstriction.

380 W e are g r a t e f u l f o r the t e c h n i c a l assistance o f Ellen W i l s o n a n d J u d y P h i p p s a n d f o r the g e n e r o u s gift o f saralasin a c e t a t e f r o m Dr. A. C a s t e l l i o n , N o r w i c h P h a r m a c a l . This w o r k was s u p p o r t e d by P r o g r a m P r o j e c t G r a n t N o . H L 1 4 3 8 8 to P G S , C a r d i o v a s c u l a r R e s e a r c h F e l l o w s h i p N o . H107007 to W E H , N S F G r a n t N o . B N S 7 5 16364 to M 1 P a n d G r a n t M R I S - 7 7 3 7 . 0 3 f r o m the V e t e r a n s A d m i n i s t r a t i o n .

1 Andersson, R., Cyclic AMP as a mediator of the relaxing action of papaverine, nitroglycerine, diazoxide, and hydrazaline in intestinal and vascular smooth muscle, Acta pharmacoL (Kbh.), 32 (1973) 321-336. 2 Andersson, R., Nilsson, K., Wikberg, J., Johansson, S., Mohme-Lundholm, E. and Lundholm, L., Cyclic nucleotides and the contraction of smooth muscle. In G. I. Drummond and G. A. Robison (Eds.), Advances in Cyclic Nucleotide Research, Raven Press, New York, 1975, pp. 491-518. 3 Barrett, J. P., Ingenita, A. J. and Procita, L., Influence of the carotid sinus on centrally mediated peripheral cardiovascular effects of angiotensin II, J. Pharmacol. exp. Ther., 176 (1971) 692 700. 4 Bickerton, R. K. and Buckley, J. P., Evidence for a central mechanism in angiotensin-induced hypertension, Proc. Sac. exp. Biol. ( N. Y.), 106 (1961) 834-836. 5 Bohr, D. F., Angiotensin on vascular smooth muscle. In 1. H. Page and F. M. Bumpus (Edsd, Handbook of Experimental Pharmtwolagy, VoI. 37 Angiotensin, Springer, Berlin, 1974, pp. 425-440. 6 Buggy, J., Fisher, A. E., Hoffman, W. E., Johnson, A. K. and Phillips, M. 1., Ventricular obstruction: effect on drinking induced by intracranial injection of angiotensin, Science. 190 (1975) 72 75. 7 Corday, E., Rothenburg, S. F. and Irving, D. W., Cerebral vasospasm : a cause of cerebral stroke, Amer. J. Cardiol., 11 (1963) 66 71. 8 Dickinson, C. J. and Yu, R., Mechanisms involved in the progressive pressor response to very smail amounts of angiotensin in conscious rabbits, Circulat. Res., 21, Suppl. 2 (1967) 157 163. 9 Ferrario, C. M., Dickinson, C. J. and McCubbin, J. W., Central vasomotor stimulation by angiotensin, Clin. Sci., 39 (1970) 239-245. l0 Fuxe, K., Ganten, D., H6kfelt, T. and Borne, P., lmmunohistochemical evidence for the exislence of angiotensin I I containing nerve terminals in the brain and spinal cord of the rat, Neurosci. Lett., 2 (1976) 229-234. I 1 Goodman, t.. S. and Gilman, A., The Pharmacological Basis oJ" Therapeutics. Macmillan, New York, 1970, pp. 727-743. 12 Gurdjian, E. S. and Thomas, L. M., Cerebral vasospasm, Surg. Gnyec. Obstet., 129 (I 969) 931 948. 13 Hoffman, W. E. and Phillips, M. I., Regional study of cerebral ventricle sensitive sites to angiotensin II, Brain Research, 110 (1976) 313 330. 14 Hoffman, W. E., Phillips, M. 1., Schmid, P. G., Falcon, J. and Weet, J. F., Antidiuretic hormone release and the pressor response to central angiotensin !1 and cholinergic stimulation, Neuropharmacology, in press. 15 Keil, L. C., Summy-Long, J. and Severs, W. B., Release of vasopressin by angiotensin 11, Endocrinology, 96 (1975) 1063-1065. 16 Lowe, R. D. and Scroop, G. C., The cardiovascular response to vertebral artery infusion of angiotensin in the dog, Clin. Sci., 37 (1969) 593 603. 17 McHenry, M. C., Jaffe, M. E., Kawamura, J. and Goldberg, H. 1., Effect of papaverine on regional blood flow in local vascular disease of the brain, New Engl. J. Med., 282 (1970) 1167- 1170. 18 Mandel, M. J. and Sapirstein, L. A., Effect of angiotensin infusion on regional blood flow and regional vascular resistance in the rat, Circulat. Res., 10 (1962) 807-815. 19 Nicolaidis, S. et Fitzsimons, J. T., La d6pendance de la prise d'eau induite par l'angiotensine 11 envers la fonction vasomotrice c6r6brate locale chez le Rat, C.R. Acad. Sci. (Paris), 281 (1975) 1417-i420. 20 Phillips, M. 1. and Hoffman, W. E., Sensitive sites in the brain for blood pressure and drinking responses to angiotensin II. In J. P. Buckley and C. Ferrario (Eds.), International Symposium on the Central Actions of Angiotensin and Related Hormones, Pergamon Press, New York, 1976, in press. 21 Pliska, V. and Rychlik, 1., Determination of antidiuretic activity in the rat for structural analogues of the neurohypophysial hormones, Acta endocr., (Kbh.), 54 (1967) 129-140.

3811 22 Rosendorff, C., Lowe, R. D., Lavery, H. and Cranston, I., Cardiovascular effects of angiotensin mediated by the central nervous system of the rabbit, Cardiovasc. Res., 4 (1970) 36-43. 23 Schmid, P. G., Mayer, H. E., Mark, A. L., Heistad, D. D. and Abboud, F. M., Differences in neural control of vascular resistance in right and left heart failure, Circulation, 50 (1974) 63-71. 24 Severs, W. B., Summy-Long, J., Taylor, J. S. and Connor, J. D., A central effect of angiotensin: release of pituitary pressor material, J. Pharmacol. exp. Ther., 174 (1970) 27-34. 25 Swanson, L. W. and Sharpe, L. G., Centrally induced drinking: comparison of angiotensin lI and carbachol sensitive sites in rats, Amer. J. Physiok, 225 (1973) 566-572. 26 Yu, R. and Dickinson, C. J., Neurogenic effects of angiotensin, Lancet, 2 (1965) 1276-1277. 27 Yuk R. and Dickinson, C. J., The progressive pressor response to angiotensin in the rabbit, Arch. int. Pharmacodyn., 191 (1971) 24-36.