Tachyphylaxis of dipsogenic activity to intracerebroventricular administration of angiotensins

Brain Research, 452 (1988) 73-78 'Elsevier

73

BRE 13627

Tachyphylaxis of dipsogenic activity to intracerebroventricular administration of angiotensins Wayne S. Quirk 1, John W. Wright 1'2 and Joseph W. Harding 1'2 1Departmentof Psychology and 2Departmentof Veterinary and ComparativeAnatomy, Pharmacologyand Physiology, Washington State University, Pullman, WA 99164-4830 (U.S.A.) (Accepted 15 December 1987)

Key words: Angiotensin II; Angiotensin III; Tachyphylaxis; Drinking; Rat

Repeated intracerebroventricular (i.c.v.) administration of angiotensin II (AII) and angiotensin III (AIII) induced dipsogenic tachyphylaxis in the Sprague-Dawley rat. AIII caused a rapid suppression of drinking, whereas AII showed a progressive decline of water consumption with repeated injections. Tachyphylaxis due to the repeated i.c.v, application of AII failed to abolish subsequent drinking induced by neurotensin or carbachol, suggesting that the tachyphylaxis may be specific to the angiotensinergic system. However, AII-induced tachyphylaxis caused a complete elimination of bestatin-induced drinking which was anticipated given the likelihood that this aminopeptidase B inhibitor has its dipsogenic effect by inhibiting degradation of endogenous angiotensins. Angiotensininduced tachyphylaxis responses could not be attributed to diminished dipsogenic activity due to volemic expansion of either the cerebroventricular space or gastrointestinal tract. These results concerned with central tachyphylaxis are consistent with previous findings in the periphery and suggest that desensitization of angiotensin receptors occurs in both populations.

INTRODUCTION There is considerable evidence to support angiotensin's role in the regulation of thirst, blood pressure, salt a p p e t i t e , and vasopressin release 5-7'21. Many of these effects have been d e m o n s t r a t e d in a number of species including rat 4'26'28, gerbil 25'26, cat 1, and non-human p r i m a t e 24. Several issues concerning the receptors responsible for mediating these actions remain unresolved. F o r e x a m p l e , the central location of these angiotensin receptors is disputed, with proposed sites in the subfornical organ II, area postrema 21, and o r g a n u m vasculosum of the lamina terminalis 19'2°. Each of these sites is located outside the b l o o d - b r a i n b a r r i e r in structures comprising the circumventricular organs, and are therefore capable of monitoring changes in angiotensin levels in the blood and cerebrospinal fluid 14'23. Certain specific characteristics of the angiotensin r e c e p t o r complex are equally unclear and in need of further study. O n e of these characteristics concerns desensitization of the

receptors to r e p e a t e d application of the angiotensin ligands. In the p e r i p h e r y , a tachyphylaxis of the pressor response to angiotensin has been o b s e r v e d in vitro in the isolated guinea pig ileum 13 and rabbit aorta ~5, and in the canine renal vascular bed 2. These studies of p e r i p h e r a l tissues and responses p r o m p t e d our l a b o r a t o r y to conduct similar examinations of central angiotensin r e c e p t o r tachyphylaxis. I n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) injections of angiotensin p r o m o t e a robust and reliable drinking response in m a n y m a m m a l i a n species and for this reason it was chosen as the target behavior. In a preliminary investigation we o b s e r v e d a tachyphylaxis of the drinking response to r e p e a t e d i.c.v, injections of angiotensin II ( A I I ) and angiotensin III ( A I I I ) in rats 9. Additionally, in vitro analyses indicated a concomitant down regulation of angiotensin receptors located in a dissected tissue block consisting of hypothalamus, septum, a n t e r o v e n t r a l third ventricle and thalamus 9. The purposes of the present investigations were to:

Correspondence: J.W. Wright, Department of Psychology, Washington State University, Pullman, WA 99164-4830, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

74 (1) confirm and extend our observations concerning central tachyphylaxis of AII and AIII-induced water consumption; (2) evaluate the influence of this angiotensin-induced tachyphylaxis on other centrally active dipsogens including carbachol, neurotensin, and bestatin; and (3) ensure that the pattern of decreased responding was not due to a behavioral deficit caused by volemic expansion of the cerebroventricular space or the gastrointestinal tract

MATERIALS AND METHODS Adult male Charles-River-derived SpragueDawley rats (300-350 g) were maintained in individual cages at 20-22 °C on a 12-h photoperiod, initiated at 07.00 h for a minimum of 7 days prior to testing. Water and Purina Lab pellets were available ad libitum except during testing. The animals were each prepared with a chronic i.c.v, guide cannula (PE 60, Clay Adams) stereotaxically positioned above the left lateral ventricle. Flat skull coordinates were 1.0 mm posterior to bregma and 1.5 mm lateral from midline. A heat-produced bubble in the guide cannula 2.5 mm above the beveled tip served as a stop for penetration depth below the top of the cranium 27. The cannula was secured to the skull with two machine screws and dental acrylic. Surgery was performed under Equithesin anesthesia (3.0 ml/kg, Jensen-Salsbury) and each animal received a prophylactic injection of procain penicillin G (0.1 ml i.m., 200,000 U/ml, Combiotic, Pfizer, NY). The animals were allowed 48 h recovery and then pretested with a bolus injection of All at a dose of 10 pmol in a total volume of 2 gl artificial cerebrospinal fluid (ACSF) 12. Pretesting was accomplished by inserting a preloaded injector into the guide cannula. The injector was fashioned from 24-gauge stainlesssteel tubing and extended 2 mm beyond the cannula tip thus penetrating the roof of the lateral ventricle. The injector was connected to a 10-gl Hamilton microsyringe by a 30 cm length of PE 20 tubing. Water consumption was measured to the nearest 0.1 ml by using 25-ml measuring tubes prepared with stainlesssteel ballbearing spouts. If a robust drinking response was not observed within the first 5 rain following the injection the animal was replaced. Animals

were allowed two days recovery before the initiation of the experimental protocols. The placement of the guide cannula was verified at post-mortem by the presence of green dye within the ventricles following a 5-BI i.c.v injection.

Angiotensin tachyphylaxis A group of 16 rats was initially injected with ACSF (2/d) as a control for dipsogenicity in response to increased ventricular volume. Ten rain later 10 pmol of AII in 2/A ACSF was delivered into the lateral ventricle and water consumption was measured for the next 20 min. Four subsequent injections of AII (10 pmol in 2 ~tl of ACSF) were administered at 20-min intervals and water consumption following each injection was measured. A second group of 16 animals was treated equivalently, however A I I I was substituted for AII. In order to control for gastric fill, 4 animals from each group were prepared with a gastric fistula, according to the procedures described by Young et al. 29, under Equithesin anesthesia. The fistula was implanted in the rumen of the stomach through a midline incision and secured with silk suture and marlex mesh (Bard Implants Division). The fistula was exteriorized through the lower flank and secured with a threaded collar loosely tightened onto the fistula shaft. The fistula was uncapped prior to testing and the consumed water flowed from the stomach via the fistula. The animals were allowed a minimum of 3 days recovery from surgery prior to testing.

Effects of other dipsogens following angiotensin-induced tachyphylaxis Three additional groups of 8 animals each were used to test for the influence of angiotensin-induced tachyphylaxis on drinking induced by other centrally active dipsogens. The previously described protocol was used with 5 injections of AII (10 pmol in 2 ~1 of ACSF) followed by a 6th injection 20 min later of neurotensin (100 nmol in 2 ,ul of ACSF), carbachol (100 nmol in 2~1 of ACSF) or bestatin (100 nmol in 2 /zl of ACSF). Drinking in response to each dipsogen following angiotensin-induced tachyphylaxis was compared to water consumption induced by each dipsogen in independent trials conducted on separate days in the same animals.

75

Behavioral deficit analysis Two final groups of 8 animals each were used to investigate the possibility that the tachyphylaxis response was due to a behavioral deficit caused by cerebroventricular expansion. Members of one group received an initial injection of 2/~1 of A C S F followed by 5 subsequent injections of 2/~1 A C S F at 20 min intervals. The animals in the second group received 4 injections of 2 fll of A C S F and a final injection of A l l (10 pmol in 2 ul ACSF). Water consumption following this last injection of A l l was compared to A l l drinking induced by the first injection in the initial experiment in order to ensure that volemic expansion, or repeated injections, did not reduce water intake induced by 10 pmol of All.

Statistical analyses Water consumption during test intervals was analyzed by repeated measures analysis of variance (ANOVA). Repeated measures A N O V A s were also used to analyze water intake of fistulated and non-fistulated animals A priori established two-tailed t-tests were used to further evaluate for differences in water consumption to carbachol, neurotensin and bestatin in independent trials and following angiotensin-induced tachyphylaxis. Additionally, t-tests were used to analyze mean water consumption of the first injection of A I I in the initial experiment with water consumption following the last injection of A I I in the volemic behavioral deficit experiment.

consumption decreased with repeated injections of angiotensin. There was also an overall difference between the treatment groups, (F = 6.46, df = 2,37, P < 0.005), and a significant change in water consumption following the initial injection of A I I or A I I I (F = 19.80, df = 5,184, P = 0.001). A priori established ttests indicated no significant difference between the water consumption of the angiotensin groups following the 10-min injection, however, following the 30min injection the group that received A I I I drank significantly less than those that received A I I (t = 2.48, df = 30, P < 0.02). The difference in patterns of tachyphylaxis response resulted in greater total water consumption by the group treated with A I I (:~ = 6.6 ml) as compared with those administered A I I I (,~ = 3.6 ml; t = 1.72, df = 30, P < 0.05; Table I). Animals prepared with gastric fistulas responded equivalently to non-fistulated animals in both groups receiving repeated injections of A I I (F = 0.64, df = 1,14, P > 0.10) or A I I I (F = 0.10, df = 1,14, P > 0.10) (Table I). Those animals that received repeated i.c.v, injections of A C S F failed to drink any water.

Effects of other dipsogens following angiotensin-induced tachyphylaxis The water consumption induced by each dipsogen following angiotensin-induced tachyphylaxis is pre-

0

E

RESULTS

e-

Angiotensin tachyphylaxis

E

Water intake (ml/20 min) due to the initial i.c.v, injection of 2 ~1 A C S F followed by repeated injections of A I I or A I I I are presented in Fig. 1. The initial injection of A C S F failed to reliably induce water consumption, however the initial injection of A I I or A I I I elicited robust dipsogenic responses. Subsequent injections of A I I showed a progressive decrease in responsiveness, while subsequent injections of A I I I were characterized by a more rapid desensitization, with a mean water intake of only 0.19 ml following the second injection. The statistical analyses supporting these conclusions indicated a significant interaction between treatments and time of injection (F = 2.86, df = 10,184, P < 0.005), indicating that water

to

o

4.0

¢~ g

,.0 c c.~ "3 "3 a . c ~w

c

c~ i

CSF

,b

3'0 go

7b

9'0

Time of Injection (min)

Fig. 1. Mean (+ S.E.M.) water consumption (ml) following i.c.v, injections of AII or AIII. Sixteen animals received an initial injection of 2 BI of ACSF and 5 subsequent injections of AII (10 pmol in 2gl of ACSF). Another 16 animals received an initial injection of 2 Itl of ACSF and 5 subsequent injections of AIII ( 10 pmol in 2 ul of ACSF). An additional volemic control group of 8 rats received an initial injection of 2 BI of ACSF followed by 5 subsequent injections of 2,ul ACSF.

76 TABLE I Mean + S.E.M. o f water consumption o f fistulated and non-fistulated Sprague-Dawley rats following repeated injections of A l l (10 pmol in 2 Bl A CSF) or A 111 (10 pmol in 2 Bl A CSF) n

A CSF

10 Min

30 Min

50 Min

70 Min

90 Min

Total

AI! Fistulated Non-fistulated Total

4 12 16

0.23 + 0.15 0.08 _+0.11

3.40 + 1.41 4.15 + 1.19 3.96 + 0.94

0.88 ± 0.43 1.59 _+0.59 1.41 _+0.43

0.58 _+0.43 0.44 _+0.33

0.33 + 0.23 0.25 + 0.18

0.42 +_0.26 0.31 _+0.26

4.28 7.30 6.55

AIII Fistulated Non-fistulated Total

4 12 16

0.13 _+0.13 0.05 + 0.07

3.25 + 0.78 2.93 + 0.70 3.00 _+0.54

0.25 +_0.25 0.38 + 0.26 0.28 + 0.19

0.18 _+0.18 0.14 + 0.14

0.13 _+0.13 0.10 _+_+0.10

sented in Table II, along with water consumption following the i n d e p e n d e n t trials conducted on separate days. t-Tests indicated no significant difference between the water c o n s u m p t i o n m e a s u r e d during the independent trials and following angiotensin-induced tachyphylaxis in response to carbachol (t = 0.62, df -7, P > 0.10). T h e r e was a significant difference between water consumption induced by neurotensin following angiotensin-induced tachyphylaxis and independent testing (t = 2.60, df = 7, P < 0.05) indicating that angiotensin-induced tachyphylaxis somewhat suppressed water consumption in response to neurotensin but did not eliminate it. It is conceivable that the previous water c o n s u m p t i o n to the r e p e a t e d injections of A l l may have had a greater impact on subsequent neurotensin-induced drinking than carbachol, although the volumes consumed in the independent trials were statistically equivalent (t = 0.19, df = 14, P > 0.10). A n g i o t e n s i n - i n d u c e d tachyphylaxis completely suppressed bestatin-induced drinking and thus yielded significant differences as compared with the i n d e p e n d e n t trials (t = 6.47, df = 7, P < 0.001). TABLE II Mean + S. E.M. of water consumption in response to intracerebroventricular injections of carbachol, neurotensin and bestatin in independent trials and following angiotensin-induced tachyphylaxis n

Carbachol (100 nmol) 8 Neurotensin (100 nmol) 8 Bestatin (100 nmol) 8

Independent trial

Post tachyphylaxis

5.54 _+2.08 5.12 + 0.76 6.04 + 0.87

4.20 + 0.98 3.04 + 1.84 0

3.66 3.49 3.57

B e h a v i o r a l deficit analysis

The final group of animals showed no dipsogenic activity to r e p e a t e d injections of A C S F , while the mean water intake following the subsequent A I I injection was 4.81 ml. This intake was not significantly different from the mean water intake (3.96 ml) following the initial injection of A I I in the first experiment (t = 0.71, df = 22, P > 0.10), indicating that the observed tachyphylaxis was not due to a b e h a v i o r a l deficit caused by r e p e a t e d injections into the lateral ventricle or by cerebroventricular expansion. DISCUSSION The results of this investigation indicate that central tachyphylaxis of angiotensin-induced water consumption does occur, and the p a t t e r n is c o m p a r a b l e with previous findings in the p e r i p h e r y ~6"~7, h o w e v e r A I I I p r o m o t e s a m o r e rapid desensitization than A l l . This finding implies that A I I I may be m o r e p o t e n t than A I I at the angiotensin receptor. This i n t e r p r e t a tion is consistent with recent evidence that A I I I has a 3 times higher absolute potency than A l I according to direct quantitative comparison of i o n t o p h o r e t i c a l ly applied A l l and A l l I in the p a r a v e n t r i c u l a r nucleus of the h y p o t h a l a m u s of rats 8. A d d i t i o n a l l y , low i.c.v, doses of A I I and A I I I are e q u i p o t e n t with respect to both pressor and dipsogenic activity in rats 26'27, despite the fact that A I I I is m e t a b o l i z e d at 3 times the rate of A I I in the c e r e b r o v e n t r i c u l a r space 1°. The neurotensin and carbachol results indicate that the tachyphylaxis resulting from angiotensin did not block drinking to o t h e r dipsogens although it did

77 suppress subsequent neurotensin-induced water consumption. This is consistent with multiple neural mechanisms regulating water intake. The failure of bestatin to induce drinking following angiotensin tachyphylaxis was anticipated since this aminopeptidase B inhibitor has recently been shown to interact with the angiotensin receptor-peptidase complex 2s and is proposed to have its dipsogenic effect by inhibiting the degradation of endogenous angiotensins ~s. Taken as a whole, these results suggest that repeated application of All or AIII may modify the receptor-transducer complex such that desensitization occurs. The potential changes occurring at the angiotensin receptor during tachyphylaxis include progressively slower receptor off-rates, receptor down regulation and internalization, and the uncoupling of the receptor and transduction system. No definitive study of angiotensin receptor binding of appropriate brain regions in desensitized animals has been completed. Chronic i.c.v, administration of AII via osmotic pump has been shown to result in no measurable differences in [~25I]AII binding site density or binding affinity in the hypothalamus-thalamus-septum-midbrain taken as a block, or the brainstem as compared with saline-treated control rats 22. An initial substantial increase in water consumption with AII infusion during the first 3 days was followed by a significant decline during the last 3 days of infusion. This drinking pattern has also been reported by DiNicolantonio et al. 3 using a similar protocol, leading these investigators to suggest that the decline may be due to down regulation of central All receptors. Although the binding results by Singh et al. 22 weigh against the possibility of down regulation, the present pattern of tachyphylaxis to repeated injections of angiotensins, and the previously reported declines in drinking over days of chronic infusion, sup-

port this notion. In addition, the protocol utilized by Singh et al. may have masked receptor changes because the tissue block was very large and primarily contained receptors from structures distant from the ventricles and therefore inaccessible to i.c.v, applied angiotensins. An in vitro binding preparation in which the hypothalamus, septum, anterior third ventricle, and thalamus were dissected out, homogenized and pre-incubated for 30 min with All or AIII indicated significant reductions in [125I]-AII binding 9. These results support the notion that if the ligand is allowed to interact with the receptors, down regulation and perhaps internalization do occur. It would therefore appear that the possibility of in vivo alterations in angiotensin receptor characteristics with chronic infusion or repeated injections has not been adequately investigated and will require additional attention. In summary, these findings support the following conclusions: (1) central tachyphylaxis of All and AllI-induced water consumption does occur with a somewhat more rapid desensitization to AIII; (2) the tachyphylaxis response may be specific to the angiotensin system since post-tachyphylaxis drinking was seen,to carbachol and neurotensin but not to bestatin; (3) the decrease in responding was not due to a behavioral deficit caused by volemic changes in the ventricles or the gastrointestinal tract.

ACKNOWLEDGEMENTS Thanks are due Dr. Bob Ritter, Washington State University for teaching us the gastric fistula preparation, and Mrs. Ruth Day for excellent secretarial assistance. Supported by NIH Grant RO-HL32063, the American Heart Association and its Washington Affiliate.

REFERENCES 1 Cooling, M.J. and Day, M.D., Inhibition of renin-angiotensin-induced drinking in the cat by enzyme inhibitors and by analogue antagonists of angiotensin II, Clin. Exp. Pharmacol. Physiol., (1974) 389-396. 2 DeLeeuw, P.W., Meggs, L.G. and Hollenberg, N.K., Renal vascular tacbyphylaxis to angiotensin II: specificity of the response for angiotensin, Life Sci., 30 (1982) 813-819. 3 DiNicolantonio, R., Mendelsohn, F.A.O., Hutchinson, J.S., Takata, Y. and Doyle, A.E., Dissociation of dipso-

4 5 6 7

genic and pressor responses to chronic central angiotensin in rats, Am. J. Physiol., 242 (1982) 498-504. Epstein, A.N., Fitzsimons, J.T. and Rolls, B.J., Drinking induced by angiotensin in the brain of the rat, J. Physiol., (Lond.), 210 (1970) 457-474. Fitzsimons, J.T., Angiotensin, thirst, and sodium appetite: retrospect and prospect, Fed. Proc., 37 (1978) 2669-2675. Fitzsimons, J.T., Angiotensin stimulation of the nervous system, Rev. Physiol. Biochem. PharmacoL, 87 (1980) 117-167. Ganong, W.F., The brain renin-angiotensin system, Ann.

78

Rev. Physiol., 46 (1984) 17-31. 8 Harding, J.W. and Felix, D., Angiotensin sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III, Brain Research, 410 (1987) 130-134. 9 Harding, J.W., Erickson, J.B., Wright, J.W., Camera, C.G. and Abhold, R.H., In vitro down regulation and possible internalization of central angiotensin receptors. In G. DeCaro, A.N. Epstein and M. Massi (Eds.), Thirst and Sodium Appetite, Plenum, New York, 1986, pp. 333-338. 10 Harding, J.W., Yoshida, M.S., Dilts, R.P., Woods, T.M. and Wright, J.W., Cerebroventricular and intravascular metabolism of [125I]angiotensin in rat, J. Neurochem., 48 (1986) 1292-1297. 11 Lind, W.R., Thornhorst, R.L. and Johnson, A.K., The subfornical organ and the integration of multiple factors in thirst, Physiol. Behav., 32 (1984) 68-74. 12 Malvin, R.L., Mouw, D. and Vander, A.J., Angiotensin: physiological role in water-deprivation-induced thirst o f rats, Science, 197 (1977) 171-173. 13 Miasaro, N., Oshiro, M.E.M., Paiva, T.B. and Paiva, A.C.M., Role of the two n-terminal residues of angiotensin II in the production of tachyphylaxis, Eur. J. Pharrnacol., 87 (1983) 387-406. 14 Mesteres, P., Old and new concepts about circumventricular organs: an overview, Scan. Electronmicrosc., 2 (1978) 137-142. 15 Oshiro, M.E.M., Miasaro, N., Paiva, T.B. and Paiva, A.C.M., Angiotensin tachyphylaxis in the isolated rabbit aorta, Blood Vessels, 21 (1984) 72-79. 16 Peach, M.J., Renin-angiotensin system: biochemistry and mechanism of action, Pharrnacol. Rev., 57 (1977) 313-370. 17 Peach, M J . , Actions of angiotensin on elements of the vascular wall and myocardium. In J.W. Harding, J.W. Wright, R.C. Speth and C.D. Barnes (Eds.), Angiotensin and Blood Pressure Regulation, Academic, Orlando, in press. 18 Quirk, W.S., Wright, J.W. and Harding, J.W., Amastatin and bestatin-induced dipsogenicity in the Sprague-Dawley rat, Brain Res. Bull., 19 (1987) 145-147.

19 Phillips, M.I., Quinlan, J., Keyser, C. and Phipps, J., Organum vasculosum of the lamina terminalis as a receptor site for ADH release, drinking, and blood pressure responses to angiotensin II, Fed. Proc., 37 (1978) 438. 20 Simpson, J-.B-., Epstein, A.N., and Camardo, Jr., J.S. Localization of receptors for dipsogenic action of angiotensin II in the subfornical organ of the rat, J. Comp. Physiol. Psychol., 92 (1978) 581-608. 21 Simpson, J.B., Mangiapane, M.1. and Dellman, H.D., Central receptor sites for angiotensin-induced drinking: a critical review, Fed. Proc., 37 (1978) 2676-2682. 22 Singh, R., Husian, A., Ferrario, C . M and Speth, R.C., Rat brain angiotensin II receptors: effects of intracerebroventricular angiotensin infusion, Brain Research, 303 (1984) 133-139. 23 Weindl, A., Neuroendocrine aspects of circumventricular organs. In W.F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Raven, New York, 1973, pp. 3-32. 24 Wright, J.W., Schultz, E.M. and Harding, J.W., An evaluation of dipsogenic stimuli in the African green monkey, J. Comp. Physiol. Psychol., 96 (1982) 78-88. 25 Wright, J.W., Morseth, S.L., LaCrosse, E. and Harding, J.W., Angiotensin III-induced dipsogenic and pressor responses in rodents, Behav. Neurosci., 98 (1984) 640-651. 26 Wright, J.W., Morseth, S.L., Mana, M.J., LaCrosse, E., Petersen, E.P. and Harding, J.W., Central angiotensin IIIinduced dipsogenicity in rats and gerbils, Brain Research, 295 (1984) 121-126. 27 Wright, J.W., Morseth, S.L., Abhold, R.H. and Harding, J.W., Pressor action and dipsogenicity induced by angiotensin II and III in rats, Am. ]. Physiol., 249 (1985) R514-R521. 28 Wright, J,W., Quirk, W.S., Hanesworth, J.M. and Harding, J.W., Influence of aminopeptidase inhibitors on brain angiotensin metabolism and drinking in rats, Brain Research, 441 (1988) 215-220. 29 Young, R.C., Gibbs, J., Antin, J. and Smith, G.P., Absence of satiety during sham feeding in the rat, ]. Comp. Physiol. Psychol., 87 (1974) 795-800.