Indian Journal of Experimental Biology Vol. 43, March 2005, pp. 241-246

Effect of losartan and enalapril on cognitive deficit caused by Goldblatt induced hypertension

J Srinivasan , S Jayaclev, D Kumaran, K F Haja Nazeer Ahamecl, B Suresh & M Ramanathan*

Neuropharmaco logy Laboratory, Department of Pharmacology,

J.S.S. College of Pharmacy, Ootacamund, 643 001 , India

Received 22 July 2004; revisecd 16 November 2004

The present study was designed to evaluate the learning and memory , in an altered physiological state assoc iated with increased bl ood pressure and acti vated renin angiotensin system in Wistar rats. The role of angiotensin in cognitive fun ction was assessed by treatment wi th angiotensin converting enzy me (ACE) inhibitor enalapril (2 mg/kg), angiotensin l receptor (AT( l )) antagoni st losartan (5 mg/kg) and their combination. The ex perimental renal hypertension was induced by the method of Gold blatt. Learning and memory was assessed using the radi al ann maze tes t. Acetylcholine esterase (AChE) leve ls in the pons medulla, hippocampus, striatum and frontal cortex were measured as a cholinergic marker of learning and memory. Results indicate th at in comparison to normotensive rats , renal hypertensive rats committed significantly hi gher number of errors and took more trial s and clays to learn the radi al arm maze learning and exhibited memory defi cit in the radial arm maze retrieval after two weeks of retention interval , indicati ng impaired acqui sition and memory. Treatment with enalapril , losartan and their combination attenuated the observed memory defi cits indicating a possi ble rol e of renin angiotensin system in cognitive functi on. AChE leve l was reduced in hippocampus and frontal cortex of renal hypertens ive rats which could be attributed to the observed memory deficit in hypertensive rats. It can be concluded th at, renal hypet·tensive rats had a poor acqu isition , retrieval of the learned behavior, perhaps a possible disturbance in memory consolidati on process and th at thi s state was reversed with ACE inhibitor enalapril and AT l receptor antagoni st losartan.

Keywords: Acetylcholine esterase, Angiotensin , Enal april , Learning and memory, Losartan, Renal hypertension

Hypertension has been attributed to cognitive deficits , psychomotor impairments and neural modifications 1'

2.

Several studies have shown the role of brain angiotensins in cognitive function, for instance, bilateral micro-injection of angiotensin II in the hippocampal area improved learning in the shuttle box3 and food finding tn the T-maze4

. Intra cerebroventricular (icv) administration of angiotensin II (2-Si, angiotensin II (3-8)6 and angiotensin II (3-7)7 exhibited memory enhancing effects 111

aversively motivated learning. However, contradictory findin gs have also been reported8

·9

. The reports on cognitive effects of antihypertensive drugs like aclrenoceptor blockers, calcium channel blockers, ang iotensin converting enzyme (ACE) inhibitors and angiotensin receptor I (AT I) antagoni sts are contradictory 111 animals and in hyperten sive subj ects 10

'11

. An increased activation of brain cholinergic 12 and adrenergic system 13

'14

111 the

*Correspondent auth or Phone: +91 -423-2443393: Fax: +91-423-2442937 e- mail: muthiahramanathanin @yahoo.co.in

hypertensive state has been demonstrated. These putative neurotransmitter systems were found to influence vanous behaviours including memory

l'd . d . 11 5 16 d conso 1 atiOn an memory retneva · an are reported to be modulated by the angiotensin system 17

The reports of ACE inhibitors and AT (I) antagonists in cognitive function in the hypertensive state were contradictory and most of the experiments were performed either in normal animals or with ic v administration of angiotensin II in normotensive rats (NTR). Hence, the present study has been planned to evaluate the cogmtJVe effects in an altered physiological state, associated with increased BP and activated renin angiotensin aldosterone system (RAAS). Since cholinergic dysfunction is correlated to cognitive impairments, AChE level was measured in different brain regions of NTR and renal hypertensive rats (RHR).

Materials and Methods

Animals - Inbred Wistar strain male Albino rats weighing 200-250 g and procured from the Central Animal House of the Institute were used. They were

242 INDIAN J EXP BIOL, MARCH 2005

housed in groups of 5-6 in colony cages at an ambient temperature of25° ± 2°C and 45-55% RH with 12:12 hr L: D cycle. They had free access to pellet chow (Brook Bond, Lipton India) and water ad libitum. Animals were exposed only once to the experiments and the experiments were performed between 0900 to 1700 hrs. The project was approved by the Institutional Animal Ethical Committee under the regul ation of CPCSEA, New Delhi.

Surgical procedure- Experimental hypertension was induced in non-fasted rats by renal occlusion method of Goldblatt et al. 18

. Surgical anesthesia was induced by chloral hydrate (400 mg/kg, ip). Lumbar surgery took place in an aseptic condition, whereby the left kidney was exposed and the branch of renal artery, suppl ying its anterior portion was occluded with aneurysm clips. Muscle and ski n were sutured separately and the rats were housed individually , until they recovered from the surgical wound. Rats were given amoxycillin (10 mg/kg) and ibuprofen (100 mg/kg) for 5 days, as a postoperative managemen t once daily. After recovery, the animals were agai n housed in colony cages for acclimatization. A similar surgery was done to the control rats omitting the occlusion step with aneurysms clips. Systoli c BP measurements were made by using tai l-cuff blood pressure recorder (IITC INC Life Science Instrumen ts) in conscious animals before the behavioral testing.

Behavioral studies

Locomotor activity - Rats were placed indiv i­dually in photoactometer, a 6 x 60 em bl ack metal chamber with a screen fl oor and a light-tight lid . Rats were placed in the chamber and allowed to acclimate fo r 2 min, then light beam breaks were counted for the nex t 5 min .

Radial arm 111aze 19 - The effect of renal hyper­

tension on learning and memory was evaluated in an eight-arm maze made of wood and elevated 50 em from the floor. The arms, each 80 em long and I 0 em wide were extended from an octagonal central platform, 35 em across . Food cups (1 em deep) were centered 2 em from the end of each arm. The testing room contained many extra maze cues and was dimly lit while sessions were in progress. Initially, the animals were given drug free training for three clays, for I 0 min . On day 4, the rats were tested one session per day, all the eight arms were baited with 1 pellet of food each . Rats were allowed to move free ly in the

maze until it collected all the 8 pellets of food or I 0 min time elapsed, whichever occurred first. The following parameters were recorded: (i) total number of errors; i.e., re-entry into baited arms that had been already visited during the session; (ii ) total arm entries; and (iii) the number of days to learn the task. The rats were declared learnt after reaching the performance of committing one mistake on three consecutive clays. After an interval of I4 drug free days of learning, the rats were again assessed once in the radial arm maze for memory retrieval.

Biochemical estimation

Acetylcholine esterase -Rats were sacrificed on the immediate day after the experiments. The brain was rapidly removed and the regions of frontal cortex, hippocampus, striatum and pons medull a were di ssected out. The ti ssue samples were homogenized (approximatel y 20 mg of the tissue per ml of phosphate buffer, pH 8, 0.1 Af) in a hand homogenizer. An aliquot (0.4 ml) of the homogenate was taken and mixed with 2.6 ml of phosphate buffer. To this, 100 fll of 5-5'-dithiobis-2- ni trobenzoic acid (Ellman's reagent; 1 mM in phosphate buffer pH 8) reagent was added and the change in absorbance was observed spectrophotometric ;> " J at 412 nm every minute after the addition of 20 fll of the substrate acetylth iocholine iodide (0.0 1 mM). The enzyme activity was expressed as mmol/min/g ti ssue10

.

The experimental design is given in Fig. 1.

Drugs - The drugs enalapril (EPL, 2 mg/kg; ENACE, Nicholas Piramal, Bombay, In dia), losartan (LSN, 5 mg/kg; LOSAR, Unisearch Laboratories, Bombay, Indi a) and their combination (ena lapril + losartan, 2+5mg/kg) were suspended in 0.3% carboxy methyl cellulose and admini stered th rough per oral route once daily and experiments were performed 45 min after drug administration.

Surgery 11.1 Acllvity box

+

Fig. 1- Schematic d iagram represents the experimental des ign

SRINIVASAN et al.: LOSARTAN AND ENALAPRIL IN COGNITION 243

Statistical analysis -Data are expressed as mean ± SE. The data on locomotor activity were subjected to Kruskal-Wallis one-way analysis of variance (ANOV A) followed by Mann-Whitney U test. Other behavioural and biochemical data were subjected to one-way ANOV A followed by Newman Keuls multiple comparison post hoc test, using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, California, USA). A P value of< 0.05 has been taken as significant.

Results

Blood pressure - Renal artery occlusion in rats significantly elevated the BP (194.0 ± 1.6 mm/Hg) in comparison to NTR (118.0 ± 0.4 mm/Hg). Treatment with EPL (2 mg/kg) (141.0 ± 0.8 mm/Hg), LSN (5 mg/kg) (147.0 ± 0.64 mm/Hg) and combination of EPL and LSN (2+5 mg/kg) (135.0 ± 0.85 mm/Hg), [F (4,25) = 9.5 I, p<0.05], significantly lowered the BP. In NTR, no effect on BP was observed with these drugs.

Locomotor activity - Renal hypertensive rats showed increased spontaneous locomotor behavior in comparison to sham controls. Treatment with EPL, LSN and their combination attenuated (X2 = 12.35,

25 .-------------------------------.

c=:J Vehicle 20 ~ EPL

~ LSN

0IIID EPLSN

c 15 ~ <f) Q)

0 0

(f) 10

NTR RHR Fig. 2-- Effect of enalapril (EPL) and losartan (LSN) and its combination on spontaneous motor activity in normotensive and renal hypertensive rats [Values are mean ± SE from 6 animals in each group. p values *< 0.01 in comparison to respective vehicle treated groups; **<0.01 in comparison to respective normotensive groups (Kruskal-Wallis one-way AN OVA followed by Mann Whitney U test) EPLSN: enalapril + losartan, NTR: normotensive rats, RHR: renal hypertensive rats]

p<O.Ol) the observed increased locomotor activity in RHR. No effect on locomotor activity was observed in NTR with these drugs (Fig. 2).

Radial arm maze learning - RHR learned the radial arm maze, with increased number of trials , more days, and committed more errors in comparison to normotensive vehicle treated rats . EPL, LSN and their combination treated RHR took less trials [F (4,25) = 14.92, p<0.01], committed less errors [F (4,25) = 9.04, p<0.05] to learn the radial arm maze in comparison to hypertensive vehicle treated groups. However, the number of days to learn the task remained unchanged in comparison to vehicle treated RHR (Table 1). Further, the drug treated RHR differed in their learning pattern in comparison to the

Table I - Effect of enalapril (EPL) and losartan (LSN) and its combination on radi al arm maze learning and relearning in

normotensive and renal hypertensive rats

[Values are mean± SE from 6 animals in each group)

Treatment Trials/day Errors/day Days

Learning

NTR (vehicle) 10.43 ± 0.28 2.43 ± 0.30 6.0 ± 0.28

EPL (2 mg/kg) 10.30 ± 0.82 2.30 ± 0.43 6.4 ± 0.4

LSN (5 mg/kg) 12.00 ± 0.56 3.90 ± 0.39 6.5 ± 0.8

EPL + LSN 10.87 ± 0.35 2.87 ± 0.31 7.2 ± 0.4 (2 + 5mg/kg)

RHR (vehicle) 13.48 1± 0.88 5.1 5 1±0.56 10.0 1± 0.49

EPL (2 mg/kg) 11.65* ± 0.61 3.55* ± 0.43 11.0 ± 1.0

LSN (5 mg/kg) 11.0 I* ± 0.68 2.81 * ± 0.68 10.5 ± 1.2

EPL+ LSN 11.03** ± 0.51 2.73* ± 0.27 12.0 ± 0.62 (2 + 5mg/kg)

Retrieval Trials Errors Days (after 14 days)

NTR (Vehicle) 8.66 ± 0.56 0.66 ± 0.3

EPL (2 mg/kg) 8.43 ± 0.75 0.43 ± 0.2

LSN (5 mg/kg) 8.73 ± 0.61 0.73 ± 0.3

EPL+ LSN 8.70 ± 0.65 0.70 ± 0.2 (2 + 5 mg/kg)

RHR (vehicle) 10.81t ± 0.30 2.81 1± 0.6

EPL (2 mg/kg) 8.40* ± 0.59 0.40* ± 0.5

LSN (5 mg/kg) 8.62* ± 0.58 0.62* ± 0.5

EPL+ LSN 8.50* ± 0.73 0.50* ± 0.2 (2 + 5 mg/kg)

P values: *< 0.05 and **< 0.01 versus respective vehicle-treated groups; t< 0.05 and 1<0.01 versus respective normotensive groups; (One way ANOV A followed by Newman Keuls test).

NTR: normotensive rats, RHR: renal hypertensive rats

244 INDIAN J EXP BIOL, MARCH 2005

respective control group indicating altered learning of hypertensive rats with drugs' treatment (Fig. 3).

Radial arm maze retrieval - In comparison to NTR, RHR committed more errors and taken more trials in radi al arm maze retri eval, 14 days after learning. However, EPL (2 mg/kg), LSN (5 mg/kg) and LSN + EPL treated hypertensive rats committed less errors [F (4,25) = 5.66, p<0.05] and took Jess number of trial s (F (4,25) = 6.12, p<0.05) in

20~------------------------------------.

-- NTRV -o- NEPL ~ NLSN

15 -6- NEPLSN

-- RHRV -o- REPL

-- RLSN ~ -o- REPLSN e 10 w

6

Number of errors in every 20% tria ls

Fig. 3- Effect of enalapril (EPL) and losanan (LSN) and its co mbinat ion on learn ing graph (errors committed in every 20% tria ls) of renal hypertensive and normotensive rats in rad ial ann maze performance. I Va lues are mea n ± SE from 6 animal s in each group NTRV: normotensive vehi cle. NEPL: normotensi ve enalapr·ii, NLSN : normotensive losar·tan , NEPLSN: normotensi ve ena lapril + losarta n. RHRV: hypertensive ve hi cle, REPL: hypertensive enalapr·ii, RLSN: hypertensive losartan, REPLSN: hypertensive enalapril + losartan]

comparison to vehicle treated group. Retrieval of normotensive rats with the treatment of EPL, LSN and their combination remains unaltered (Table I).

Biochemical estimation

Acetylcholine esterase - Experimentally induced renal hypertension significantly decreased the AChE concentration in the hippocampus and frontal cortex reg ions in comparison to NTR. LSN alone and its combination with EPL increased the AChE level in the hippocampus [F (4,25) = 24.86, p<O.Ol] and cortical regions [F (4,25) = 137.7, p<0.001] of both NTR and RHR in compari son to their respecti ve vehicle treated groups . Further, LSN also elevated the AChE level in pons medulla [F (4,25 ) = 5.87, p<0.05] and stri atal region s [F (4,25) = 6.1 5, p <0.05] of RHR. EPL (2 mg/kg) did not alter the AChE level in hypertensive rats. In NTR, EPL increased the hippocampal (F (4,25) = 28.79, p<O.OO I) and cortical (F (4,25) 79 .32, p<O.OO I) AChE level 111

compari son to vehicle treated normotensive rats. Similar findings were observed w ith combinati on treatment (Table 2).

Discussion

The res ults of the prese" ' study indicate that experimen tal hyperten sion in ra ts produced acqui s ition and memory defi c it as indicated by inet·eased trial s, working me mory errors in the rad ia l arm maze learning and retri eva l. Treatment with ACE inhibitor EPL, AT(l) antago nist LSN and the ir combination reversed the observed me mory and acquisition deficit in RHR. These resu lts indicate that the blockade of angiotensin may have benefici al

Ta ble 2 - Effecl of enalapril (EPL) and losartan (LS N) and its combination on brain acetylchol ine esterase level (mmols/min/g tissue) in normotensive and renal hypertensive rats

(Va lues are mean± SE from 6 animals from each group]

Groups Pons medulla Hippoca mpu s Striatum Frontal cortex

NTR (vehi cle) 4.35 ± 0.12 4.86 ± 0.05 6.01±0.14 7.1 7 ±0.27

EPL (2 mg/kg) 3.96 ± 0.09 6.06 ± 0.04 5.95 ± 0.30 8.44* ± 0.26

LSN (5 mg/kg) 4.77± 0.21 5.57* ± 0.29 6.54 ± 0.04 X.03*± 0.15

EPL + LSN (2 + 5 mg/kg) 4. 87 ± 0. 35 7.63* ± 0.56 6. 33 ± 0.06 8.68* ± 0.43

RHR (vehicle) 3.97 ± 0.04 3.261± 0.1 8 5.40 ± 0.20 3.431 ± 0.21

EPL (2 mg/kg) 4.53 ± 0.26 3.941 ± 0.09 5. ll t ±0. 15 3.60 1 ± 0.22

LSN (5 mg/kg) 4.70' ± 0.29 4.06''' ' ± 0. 10 6.33* ± 0.24 4.01 *1±0.06

EPL + LSN (2 + 5 mg/kg) 4.05 ± 0.50 6.68* ± 0.23 5.68 ± 0.69 5.06* ± 0.08

P Va lu es:*< 0.05 versus respective vehi cle lreatecl groups; t< 0.05 anc! 1< 0.0 1 versus respective normotensive groups (One way AN OVA fol lowed by Newma n Keul s test) NTR: normolensive rats; RHR: renal hypertensive rats

SRINIVASAN et at. : LOS ART AN AND ENALAPRIL IN COGNITION 245

cognitive effects in the hypertensive state. In contradiction, direct administration of angiotensin in the brain improved the cogmtive function in aversively motivated learning6

-8

. Further, bilateral microinjection of angiotensin II into the hippocampal area improved learning in the shuttle box4

. In the T­maze, angiotensin II administration increased the efficiency of food finding5

. All these reports clearly indicate the facilitatory role of angiotensin II in the learning and memory process on direct admin istration. However, contradictory findings have also been reported9

·10

. The discrepancies of the present findings in hypertensive state may be attributed to the altered physiological conditions; where the BP is elevated and RAAS is activated21

.

Hence, the memory deficit observed with RHR and its reversa l by EPL and LSN indicate the importance of RAAS in memory formation.

Hypertensive animals committed an increased number of working memory errors in the radial arm maze acquisition and failed to show the retrieval of the learned task after two weeks retention interval indicating poor acquisition and memory defici t. It may be attributed to the alteration in the long- term potentiation (L TP) pathway, as L TP is influenced by angiotensin22 resulting in poor consolidation process. Though in hypertensive state increased adrenergic and serotonergic activity have been reported 13

·14

, learning deficits in hypertensive animals were observed in the present study, indicating the role of other biochemical/neurohumoral alterations that cou ld be responsible for the observed deficit. EPL, LSN and its combination treated hypertensive rats produced better retrieval in the radi al arm maze, further confirming the ro le of angiotensin in cogniti ve function and memory consolidation . In the present study, decreased AChE concentration was observed in hypertensive rats especi all y in the hippocampal and cortical structures. Intact cholinergic actiVIty has been implicated to have a better working memory23

. Earlier reports indicate th at, ACE inhibitors and AT antagonist produced better learning and memory performance in hypertensive animals by enhancing the cholinergic activit/4 which is in accord to the present study. All these results clearly indicate that in hypertensive state there is a possible alteration in the cholinerg ic function and memory consolidation process. The possible restoration of the cholinergic activ ity with the antihypertensive drugs cou ld be attributed to its beneficial cognitive effects.

Therefore, it can be concluded that the hypertensive animals had both acquis1t10n and memory deficits of the learned behaviour in radial arm maze, indicating a possible altered memory consolidation process and it was reversed with ACE inhibitor EPL (2 mg/kg), AT(l) antagonist LSN (5 mg/kg) and the combination of EPL + LSN (2 + 5 mglkg).

References

Wilkie F & Eisdorfer C, Intelli gence and blood pressure in the aged, Scie11ce, 172 ( 1971) 959.

2 Hacioglu G, Agar A, Ozkaya G, Yargicoglu P & Gumuslu S, The effect of different hypertension models on ac ti ve avoidance learning, Brain Cognitio11, 52 (2003) 216.

3 Belcheva I, Ternianov A & Georgiev V, Lateralized learning and memory effects of angiotens in II microinjected into the rat CA I hippocampal area, Peptides, 21 (2000)'407.

4 Bmszko J J, Kulakowska A & Karwowska-Polecka W, CGP 42 11 2A antagonism of the angiotensi n II and angiotensi n II (3-7) facil itation of 1·eca ll in rats, Phan11acol. Res, 38 (1998) 461.

5 Braszko J J, Wi sniews ki K, Kupryszews ki G & Witczu k B, Psychotropi c effects of angiotensin II and III in rats: Locomotor and exploratory Vs cogn iti ve behaviour, Behav Brain Res, 25 ( 1987) 195.

6 Wri ght J W, Miller-Wing A V, Schaffer M J, Hi gginson C, Wri ght D E, Hanesworth J W & Harding J W, Angiotensi n II (3-8) (ANG IV) hippocampal binding: Potential role in the facilitation of memory. Brain Res Bull, 32 ( 1993) 497.

7 Braszko J J, Wlasienko J, Koziolkiewicz W, Janecka A & Wi sn iewski K, The 3-7 fragment of angiotensin II is probably responsible fo r its psyc hoacti ve properties, Brai11 Res, 542 ( 1991 ) 49.

8 Raghavendra V, Chopra K & Kulkarni S K, Brain renin angio tensin system (RAS) in stress- induced analges ia and impaired retention , Peptides , 20 ( 1999) 335.

9 Tchekalarova J, Pechli vanova D, Kambourova T, Matsoukas J & Georgi ev V, The effects of sannesin , an Angiotensin II analogue on seizure susceptibility, memory retention and noc iception , Regul Pep!, Ill (2003) 191.

10 Prince M J, Bird A S, Blizard R A & Mann A H, Is the cogn iti ve function of older patients affected by ant ihypertens ive treatment? Results from 54 months of Medical Research Council ' s 1rial of hypertension in older adults , BMJ, 3 12 (1996) 801.

II Bulpitt C J & Fletcher A E, Quality of life of anti hypertensive drugs. Pharmacoeco11omics, I ( 1992) 95.

12 Buccafusco J J & Spector S, Role of central cholinergic neurons in experimental hypertension, J Cardiovasc Pharmacal , 2 ( 1980) 347.

13 Yao H. Monoamine co ntents and norepinephrine turnover in brain stem nuclei of young and adult spontaneously hypertensive and Wistar Kyoto rats, Fukuka lgaku Zasshi , 8 1 ( 1990) 370.

246 INDIAN J EXP BIOL, MARCH 2005

14 Esler M, Ferrier C, Lambert G, Eisenofer G, Cox H & Jen nings G, Biochemical evidence of sympathetic hyperac ti vity in human hypertension, Hypertension, 17 (1991) 29.

15 Vi zi E S & Kiss J P, Neurochemistry and pharmacology of the major hippocampal transmitter systems: synaptic and nonsynaptic interact ions, Hippocampus, 8 ( 1998) 566.

16 Smiley J F, Monoamines and acetylcholine in primate cerebral cortex: What anatomy tell s us about function , Rev Braz Bioi, 56S l ( 1996) 153.

17 Govantes C & Marin J, Effect of angiotensin converting enzyme inhibitors on quality of life in hypertensive patients: Pharmacodynami c basis, Fund am Clin Phannacol, I 0 ( 1996) 400.

18 Goldblatt H, Lynch J, Hanzal R F & Summerville W W, Studies on ex perimental hypertension - the production of persistent evaluat ion of blood pressure by means of renal ischemia, J Exp Med, 59 ( 1934) 34 7.

19 Olton D S & Samuelson R J, Remembrance of places passed: spatial memory in rats, J Exp Psycho! Anim Behav Proc, 2 (1976) 97.

20 Ellman G L, Courtney D, Andres V & Featherstone R M, A new rapid colorimetric determination of acetylcholine esterase activity, 7 ( 1961) 88.

2 1 Jagadeesh G, Angiotensin II receptors-antagonists, molecular biology, and signal transduct ion, Indian J Exp Bioi, 36 (1998) 1171.

22 Wright J W, Kramar E A, Meighan S E & Harding J W, Extracellular matrix molecules, long-term potentiation , memory consolidation and the brain angiotensin system, Peptides, 23 (2002) 221.

23 Fadda F, Melis F & Stancampiano R, Increased hippocampal acetylcholine release during a working memory task, Eur J Pharl/lacol, 307 (1996) Rl.

24 Raghavendra V, Chopra K & Kulkarni S K, Involvement of cholinergic system in losanan-induced fac ilitation of spatial and sho11-term working memory, Neuropeptides, 32 (1998) 41 7.