Effects of Central Imidazolinergic and Alpha2-Adrenergic Activation on Water Intake

Brazilian Journal of Medical and Biological Research (2001) 34: 1185-1190 Imidazolines, a2-adrenoceptors and water intake 1185 ISSN 0100-879X

Effects of central imidazolinergic and alpha2-adrenergic activation on water intake

A.M. Sugawara, T.T. Miguel, Departamento de Fisiologia e Patologia, Faculdade de Odontologia, D.T.B. Pereira, Universidade Estadual Paulista, Araraquara, SP, Brasil J.V. Menani and L.A. De Luca Jr.

Abstract

Correspondence Non-adrenergic ligands that bind to imidazoline receptors (I-R), a Key words · L.A. De Luca Jr. selective ligand that binds to a2-adrenoceptors (a2-AR) and mixed Imidazolines Departamento de Fisiologia e ligands that bind to both receptors were tested for their action on water · Agmatine Patologia intake behavior of 24-h water-deprived rats. All drugs were injected · UK 14304 Faculdade de Odontologia, UNESP into the third cerebral ventricle. Except for agmatine (80 nmol), mixed · Guanabenz Rua Humaitá, 1680 · a ligands binding to I-R/a -AR such as guanabenz (40 nmol) and UK -Methylnoradrenaline 14801-903 Araraquara, SP 2 · 14304 (20 nmol) inhibited water intake by 65% and up to 95%, Thirst Brasil · Dehydration Fax: +55-16-201-6488 or 222-4823 respectively. The selective non-imidazoline a2-AR agonist, a-methyl- E-mail: [email protected] noradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol). The non- Research supported by CNPq- adrenergic I-R ligands histamine (160 nmol, mixed histaminergic and PRONEX and FAPESP. A.M. Sugawara imidazoline ligand) and imidazole-4-acetic acid (80 nmol, imidazoline was the recipient of a graduate fellowship from FAPESP. T.T. Miguel ligand) did not alter water intake. The results show that selective, non- a and D.T.B. Pereira were recipients imidazoline 2-AR activation suppresses water intake, and suggest of undergraduate fellowships from that the action on imidazoline sites by non-adrenergic ligands is not FAPESP and CNPq, respectively. sufficient to inhibit water intake. Part of a Master’s thesis presented by A.M. Sugawara to the Departa- mento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brazil. Introduction dine also binds to imidazoline receptors (I-R) (4). The inhibition of water intake

Clonidine, an antihypertensive drug that through activation of a2-AR has been re- Received April 13, 2000 binds to a2-adrenoceptors (a2-AR), has been cently confirmed in part by the antagonism Accepted June 5, 2001 known for many years to be a drug that acts of moxonidine, a mixed I1-R/a2-AR ligand, in the brain to inhibit water intake (for a by the selective a2-AR antagonist RX 821002 review, see Ref. 1). Early experiments sug- (5). However, moxonidine is more selective

gested that this effect of clonidine depends for I1-R than for a2-AR (6), and RX 821002, a on the central activation of a2-AR since it is although considered a selective 2-AR an- inhibited by yohimbine, an a2-AR antago- tagonist, also has an imidazoline structure nist (2,3). However, some experiments have (7), thereby binding occasionally to imid- shown that yohimbine does not fully antago- azoline sites (8). This opens the possibility

nize the antidipsogenic effect of clonidine that imidazoline activation, in addition to a2- (3). A possible explanation for this result is AR activation, also produces inhibition of

that, in addition to binding to a2-AR, cloni- water intake. An approach to test this hypo-

Braz J Med Biol Res 34(9) 2001 1186 A.M. Sugawara et al.

thesis is to screen the effects of drugs that not body weight and daily water intake were only are selective for one receptor, but, in back to pre-surgery values. this particular case, that also differ in terms of presence or absence of the imidazoline Surgery ring. Imidazole-4-acetic acid (IAA) is an inter- The animals were maintained under esting imidazole ligand to imidazoline re- tribromoethanol (Aldrich, Milwaukee, WI, ceptors, on the one hand because it does not USA) (20 mg/100 g body weight) anesthesia bind to adrenoceptors and on the other throughout surgery. A stainless steel guide hand, because it is produced endogenously cannula (15 x 0.7 mm OD) was stereotaxi- as a metabolite of another imidazole, hista- cally implanted into the third cerebral ven- mine (9). Histamine in turn also binds to I-R tricle (3rdV). Coordinates were AP = 0.2 (9). mm behind bregma, V = 7.6 mm from cra- Another endogenous ligand to I-R, but nial surface, L = 1.2 mm from lambda, inci-

also to a2-AR, is agmatine (decarboxylated sor bar = 2.5 mm below the interaural line, o arginine) (10). Mixed I-R/a2-AR ligands of and cannula at 10 angle from the sagittal interest (7) like guanabenz (GBZ), a guani- plane. The cannula was secured to the top of dine, and UK 14304, an imidazoline, inhibit the skull with acrylic cement and fastened sodium intake (11). Imidazolidine mixed with two screws. Insertion of a close-fitting

ligands to I1-R/a2-AR, such as clonidine and stylet kept the lumen free of debris and moxonidine, are known to inhibit both water clots. A prophylactic pre-surgical dose of and sodium intake (1-3,5,12). penicillin (30,000 IU) was given intramus- The effect of a selective non-imidazoline cularly.

a2-AR ligand has not been tested on the behavior of water intake. A useful tool for Intracerebral injections

this task is the a2-AR agonist a-methyl- noradrenaline (mNOR), which is likely to Single-pulse intracranial injections were have the same inhibitory effect on water made after gently removing the animal from intake as noradrenaline (nonselective a- and its cage, replacing the stylet with an injector ß-AR ligand) does (13). Thus, in the present that protruded 1.0 mm beyond the tip of the study we tested the effects of intracerebro- guide cannula and that was connected by ventricular (icv) injections of IAA, hista- PE-10 tubing to a 10-µl syringe, and inject- mine, agmatine, GBZ, UK 14304 and mNOR ing a total volume of 1.0 µl over a period of on water intake by water-deprived rats. 20 s. Stylet and injector were always wiped with cotton soaked in 70% alcohol between Material and Methods injections. After the injection, the injector was removed and replaced with the stylet, Animals and the animal was returned to its cage for observation of its behavior. Male Holtzman rats weighing 260-300 g at the beginning of the experiments were Drugs housed individually in a room on a 12/12-h light-dark cycle beginning at 7:00 am. Stan- Agmatine was purchased from Sigma, dard Purina pellets and tap water were avail- (St. Louis, MO, USA) and IAA, histamine, able ad libitum unless otherwise stated. All GBZ, UK 14304 and mNOR were purchased experiments began between 8:00 and 9:00 from RBI (Natick, MA, USA). The drugs am at least three days after surgery, when were dissolved in 0.9% saline, but GBZ and

Braz J Med Biol Res 34(9) 2001 Imidazolines, a2-adrenoceptors and water intake 1187 mNOR were dissolved in acidified (pH = 4.6) and 160 nmol) reduced water intake from 39 0.9% saline and UK 14304 was dissolved in to 76% during the whole test, except at 15 2:1 water:propyleneglycol solution. min for 10 and 40 nmol (Figure 1). Lower doses (2.5 and 5 nmol) of mNOR had no Histology effect on water intake.

a At the end of the experiments, the ani- Injection of the mixed I-R/ 2-AR ligands GBZ, mals were deeply anesthetized with chloral UK 14304 and agmatine and water intake in hydrate (Merck, Rio de Janeiro, RJ, Brazil) water-deprived rats (20 mg/100 g body weight) and perfused with 10% formalin through the left ventricle Icv injection of GBZ reduced water in- of the heart. After fixation in 10% formalin, take from 30% (2.5 nmol) to 65% (5, 10, 20, the brains were cut with a cryo-cut micro- 40 and 80 nmol) during the whole test (Fig- tome. Transverse sections (50 µm) were an- ure 2A). alyzed by light microscopy to confirm the UK 14304 (5 and 10 nmol) reduced wa- position of the cannula in the 3rdV. ter intake by 43 and 70% from 15 to 60 min and had no effect at 120 min (Figure 2B). Statistical analysis UK 14304 (20 and 40 nmol) reduced water intake by 90-95% from 15 to 30 min and by Data are reported as means ± SEM. Two- 60-80% from 60 to 120 min (Figure 2B). way (drug and time as factors) analysis of Agmatine (80 nmol) did not alter water variance was used for comparisons between intake (Figure 2C). groups followed by the Student-Newman- Keuls test. The level of significance was set Injection of the non-adrenergic I-R ligands at P<0.05 for all tests. IAA and histamine and water intake in water-deprived rats Water intake test Icv injection of either IAA (40 and 80 Water was removed from the cage and nmol) or histamine (160 nmol) did not alter then only food remained available for 24 h. water intake in water-deprived animals (Fig- Then, food was removed prior to returning ure 3). water to the animals. Agonists or vehicle Figure 1. Cumulative water in- was injected into the 3rdV 20 min before the mNOR 20 (7) Vehicle (10) take by 24-h water-deprived rats burettes containing water became available mNOR 2.5 (7) mNOR 40 (6) after intracerebroventricular in- mNOR 5 (6) mNOR 80 (6) to the animals. Water intake was measured 20 jection of a-methylnoradrenaline mNOR 10 (6) mNOR 160 (6) for 2 h (at 0, 15, 30, 60 and 120 min) in the (mNOR; 2.5, 5, 10, 20, 40, 80 absence of food. Water and standard food and 160 nmol) or vehicle (open 15 squares). The number of animals were returned to the animals at the end of the per group is given in parenthe- water intake test. The experiments were sepa- * ses. *P<0.05 vs vehicle (Stu- dent-Newman-Keuls test). rated by 3-4-day intervals. 10 * * * Results 5

a Cumulative water intake (ml) Injection of the selective 2-AR agonist mNOR and water intake in water-deprived rats 0 0 15 30 60 120 Time (min) Icv injection of mNOR (10, 20, 40, 80

Braz J Med Biol Res 34(9) 2001 1188 A.M. Sugawara et al.

Other behaviors disappeared and the animals looked normal at lower doses. Neither GBZ nor mNOR The highest dose (40 nmol) of UK 14304 produced any observable alteration in be- produced motor deficits such as immobiliza- havior other than inhibition of water intake. tion and prostration that lasted 30 min in 20% of the animals. This effect completely Discussion

Figure 2. Cumulative water in- The mixed I-R/a2-AR ligands GBZ and take by 24-h water-deprived rats Vehicle (8) GBZ 10 (8) 20 after intracerebroventricular in- A GBZ 2.5 (6) GBZ 20 (6) UK 14304, but not agmatine, inhibited water jection of: A, guanabenz (GBZ; GBZ 5 (7) GBZ 40 (6) intake by water-deprived rats. A similar ef- GBZ 80 (6) 2.5, 5, 10, 20, 40 and 80 nmol); fect was obtained with a-mNOR, a selective B, UK 14304 (UK; 5, 10, 20 and 15 a 40 nmol), and C, agmatine 2-AR agonist. The non-adrenergic ligands * * (AGM; 80 nmol) or vehicle (open * * to I-R, IAA and histamine, did not alter squares). The number of ani- 10 water intake. mals per group is given in paren- GBZ is considered to be a selective a - theses. *P<0.05 vs vehicle (Stu- 2 dent-Newman-Keuls test). 5 AR agonist, but it also binds to I2-R (7). UK 14304 also has preference for I2-R (7), but Cumulative water intake (ml) less affinity for a2-AR than GBZ (14). Ag- 0 matine, a ligand to a -AR and to I -R, and a 0 15 30 60 120 2 1 Time (min) potential candidate as the endogenous cloni- dine-displacing substance (10), had no ef- 20 B Vehicle (6) UK 20 (6) fect on water intake. This contrasts with the UK 5 (6) UK 40 (7) UK 10 (7) potent inhibitory effect that moxonidine and a 15 clonidine, also mixed ligands to 2-AR and to I1-R, have centrally on water intake (1- 3,5,12). Different from moxonidine and clo- 10 * nidine, agmatine has no hypotensive effect * * * when injected centrally (15,16). It is still not 5 known why it is hard to obtain an effect with agmatine. Cumulative water intake (ml) IAA had no effect on water intake, sug- 0 gesting that binding to I-R and not to a -AR 0 15 30 60 120 2 Time (min) is not sufficient to inhibit water intake. This compound injected into the rostral ventrolat- eral medulla also causes hypotension attrib- 20 C Vehicle (7) AGM 80 (6) uted to activation of I-R (17). A possible involvement of IAA through GABA recep- 15 tor binding (18,19) should have altered wa- ter intake (20), but such alteration did not 10 occur in the present study. Histamine, a mixed histaminergic/imidazoline receptor ligand and a precursor of endogenous IAA (21), 5 does not bind to a2-AR and also did not alter Cumulative water intake (ml) water intake. Failure to inhibit water intake 0 by histamine can be attributed in part to its 0 15 30 60 120 dipsogenic property expressed through acti- Time (min) vation of histaminergic receptors (22), but

Braz J Med Biol Res 34(9) 2001 Imidazolines, a2-adrenoceptors and water intake 1189 no enhancement of water intake was ob- times lower than the minimum dose needed served in the present study. to obtain an effect with mNOR. On the one

The selective a2-AR ligand mNOR (14) hand, since GBZ binds weakly to I1-R, the inhibited water intake. This is the first dem- results favor the participation of a2-AR. On onstration that a non-imidazoline selective the other hand, the higher potency of GBZ ligand to a2-AR inhibits water intake. The compared to mNOR suggests that binding to effect of mNOR on water intake is similar in I-R might help. In either case, GBZ did not potency to the effect of the endogenous ligand exert full inhibition on water intake. Thus, noradrenaline (13) and this suggests an in- some kind of interaction may occur between a hibitory role for central a2-AR in the control 2-AR and I-R, particularly in the imidazo- of thirst. However, neither noradrenaline line/imidazolidine domain, that is necessary (13), mNOR or GBZ are able to fully inhibit for the full effect or potency of the agonists. water intake by water-deprived rats. This Of course, this is not the only possible expla- contrasts with the near complete inhibition nation and higher metabolic rates, for ex- of water intake by clonidine (13), moxonidine ample, could also explain the need for higher (5) and UK 14304 (present study). doses of phenylethylamines to induce inhi- The results of the present study are con- bition. Nevertheless, this hardly explains why sistent with prior evidence for an inhibitory the inhibition produced by these compounds role of a2-AR in the control of water intake never exceeds 70% in spite of the higher (2,3,5). However, a straightforward inter- doses used. pretation of the results favoring a2-AR on the one hand and ruling out I-R on the other Figure 3. Cumulative water in- is not yet possible. Consider, first, the com- 20 A take by 24-h water-deprived rats plete central antagonism of moxonidine by after intracerebroventricular in- RX 821002, a selective a -AR antagonist jection of: A, imidazole-4-acetic 2 15 acid (IAA; 40 and 80 nmol) and (5). Nevertheless, RX 821002 is an imidazo- B, histamine (HIS; 160 nmol), or line molecule which can also bind to I-R (8) vehicle (open squares). The and moxonidine is an imidazolidine selec- 10 number of animals per group is given in parentheses. tive for I -R compared to clonidine (6,14,17). 1 Vehicle (7) Second, the inhibitory potency of the drugs IAA 40 (6) 5 on water intake seems to correlate with their IAA 80 (6) molecular nature. Clonidine, moxonidine and Cumulative water intake (ml) 0 UK 14304 are imidazolidines/imidazoline 0 15 30 60 120 a that bind to 2-AR and I-R and are the most Time (min) potent inhibitors of water intake compared 20 B to the phenylethylamines noradrenaline, mNOR and phenylephrine (a1-AR agonist). To obtain the same amount of inhibition of 15 water intake achieved with imidazolidines/ imidazoline, it is necessary to increase by 10 four- to eight- fold all the doses below the maximum dose of noradrenaline (13) and Vehicle (7) mNOR. In addition, full inhibition of water 5 HIS 160 (6) intake is not achieved with maximum doses Cumulative water intake (ml) of noradrenaline (13), mNOR and GBZ. It is 0 noteworthy, however, that the minimum dose 0 15 30 60 120 Time (min) needed to obtain an effect with GBZ is four

Braz J Med Biol Res 34(9) 2001 1190 A.M. Sugawara et al.

Although evidence has pointed out dis- on receptor characterization and cloning of tinct receptor populations (16), overlapping imidazoline-binding sites (25) will also help

of a2-AR and I-R may also complicate the to clarify possible interactions with a2-AR. interpretation of the results. This is reflected by the ongoing controversy about the role of Acknowledgments a2-AR and I-R as antihypertensive drugs (16,19,23,24). However, suggestions that an The authors thank S.P. Barbosa, R.C. interaction between these receptors does oc- Queiróz, S. Foglia and A. Vítor for technical cur are increasing (7) and our results are assistance, and S.D. Malavolta for secre- somehow consistent with them. Further data tarial assistance.

References

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