Effects of central imidazolinergic and alpha 2-adrenergic activation on water intake

Non-adrenergic ligands that bind to imidazoline receptors (I-R), a selective ligand that binds to a2-adrenoceptors ( a2-AR) and mixed ligands that bind to both receptors were tested for their action on water intake behavior of 24-h water-deprived rats. All drugs were injected into the third cerebral ventricle. Except for agmatine (80 nmol), mixed ligands binding to I-R/ a2-AR such as guanabenz (40 nmol) and UK 14304 (20 nmol) inhibited water intake by 65% and up to 95%, respectively. The selective non-imidazoline a2-AR agonist, a-methylnoradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol). The nonadrenergic I-R ligands histamine (160 nmol, mixed histaminergic and imidazoline ligand) and imidazole-4-acetic acid (80 nmol, imidazoline ligand) did not alter water intake. The results show that selective, nonimidazoline a2-AR activation suppresses water intake, and suggest that the action on imidazoline sites by non-adrenergic ligands is not sufficient to inhibit water intake. Correspondence


Introduction
Clonidine, an antihypertensive drug that binds to = 2 -adrenoceptors (= 2 -AR), has been known for many years to be a drug that acts in the brain to inhibit water intake (for a review, see Ref. 1).Early experiments suggested that this effect of clonidine depends on the central activation of = 2 -AR since it is inhibited by yohimbine, an = 2 -AR antago- nist (2,3).However, some experiments have shown that yohimbine does not fully antagonize the antidipsogenic effect of clonidine (3).A possible explanation for this result is that, in addition to binding to = 2 -AR, cloni- dine also binds to imidazoline receptors (I-R) (4).The inhibition of water intake through activation of = 2 -AR has been re- cently confirmed in part by the antagonism of moxonidine, a mixed I 1 -R/= 2 -AR ligand, by the selective = 2 -AR antagonist RX 821002 (5).However, moxonidine is more selective for I 1 -R than for = 2 -AR (6), and RX 821002, although considered a selective = 2 -AR an- tagonist, also has an imidazoline structure (7), thereby binding occasionally to imidazoline sites (8).This opens the possibility that imidazoline activation, in addition to = 2 - AR activation, also produces inhibition of water intake.An approach to test this hypo-thesis is to screen the effects of drugs that not only are selective for one receptor, but, in this particular case, that also differ in terms of presence or absence of the imidazoline ring.
Imidazole-4-acetic acid (IAA) is an interesting imidazole ligand to imidazoline receptors, on the one hand because it does not bind to adrenoceptors and on the other hand, because it is produced endogenously as a metabolite of another imidazole, histamine (9).Histamine in turn also binds to I-R (9).
Another endogenous ligand to I-R, but also to = 2 -AR, is agmatine (decarboxylated arginine) (10).Mixed I-R/= 2 -AR ligands of interest (7) like guanabenz (GBZ), a guanidine, and UK 14304, an imidazoline, inhibit sodium intake (11).Imidazolidine mixed ligands to I 1 -R/= 2 -AR, such as clonidine and moxonidine, are known to inhibit both water and sodium intake (1)(2)(3)5,12).The effect of a selective non-imidazoline = 2 -AR ligand has not been tested on the behavior of water intake.A useful tool for this task is the = 2 -AR agonist =-methylnoradrenaline (mNOR), which is likely to have the same inhibitory effect on water intake as noradrenaline (nonselective =and ß-AR ligand) does (13).Thus, in the present study we tested the effects of intracerebroventricular (icv) injections of IAA, histamine, agmatine, GBZ, UK 14304 and mNOR on water intake by water-deprived rats.

Animals
Male Holtzman rats weighing 260-300 g at the beginning of the experiments were housed individually in a room on a 12/12-h light-dark cycle beginning at 7:00 am.Standard Purina pellets and tap water were available ad libitum unless otherwise stated.All experiments began between 8:00 and 9:00 am at least three days after surgery, when body weight and daily water intake were back to pre-surgery values.

Surgery
The animals were maintained under tribromoethanol (Aldrich, Milwaukee, WI, USA) (20 mg/100 g body weight) anesthesia throughout surgery.A stainless steel guide cannula (15 x 0.7 mm OD) was stereotaxically implanted into the third cerebral ventricle (3rdV).Coordinates were AP = 0.2 mm behind bregma, V = 7.6 mm from cranial surface, L = 1.2 mm from lambda, incisor bar = 2.5 mm below the interaural line, and cannula at 10 o angle from the sagittal plane.The cannula was secured to the top of the skull with acrylic cement and fastened with two screws.Insertion of a close-fitting stylet kept the lumen free of debris and clots.A prophylactic pre-surgical dose of penicillin (30,000 IU) was given intramuscularly.

Intracerebral injections
Single-pulse intracranial injections were made after gently removing the animal from its cage, replacing the stylet with an injector that protruded 1.0 mm beyond the tip of the guide cannula and that was connected by PE-10 tubing to a 10-µl syringe, and injecting a total volume of 1.0 µl over a period of 20 s.Stylet and injector were always wiped with cotton soaked in 70% alcohol between injections.After the injection, the injector was removed and replaced with the stylet, and the animal was returned to its cage for observation of its behavior.

Histology
At the end of the experiments, the animals were deeply anesthetized with chloral hydrate (Merck, Rio de Janeiro, RJ, Brazil) (20 mg/100 g body weight) and perfused with 10% formalin through the left ventricle of the heart.After fixation in 10% formalin, the brains were cut with a cryo-cut microtome.Transverse sections (50 µm) were analyzed by light microscopy to confirm the position of the cannula in the 3rdV.

Statistical analysis
Data are reported as means ± SEM.Twoway (drug and time as factors) analysis of variance was used for comparisons between groups followed by the Student-Newman-Keuls test.The level of significance was set at P<0.05 for all tests.

Water intake test
Water was removed from the cage and then only food remained available for 24 h.Then, food was removed prior to returning water to the animals.Agonists or vehicle was injected into the 3rdV 20 min before the burettes containing water became available to the animals.Water intake was measured for 2 h (at 0, 15, 30, 60 and 120 min) in the absence of food.Water and standard food were returned to the animals at the end of the water intake test.The experiments were separated by 3-4-day intervals.

Injection of the non-adrenergic I-R ligands IAA and histamine and water intake in water-deprived rats
Icv injection of either IAA (40 and 80 nmol) or histamine (160 nmol) did not alter water intake in water-deprived animals (Figure 3).disappeared and the animals looked normal at lower doses.Neither GBZ nor mNOR produced any observable alteration in behavior other than inhibition of water intake.

Discussion
The mixed I-R/= 2 -AR ligands GBZ and UK 14304, but not agmatine, inhibited water intake by water-deprived rats.A similar effect was obtained with =-mNOR, a selective = 2 -AR agonist.The non-adrenergic ligands to I-R, IAA and histamine, did not alter water intake.
GBZ is considered to be a selective = 2 - AR agonist, but it also binds to I 2 -R (7).UK 14304 also has preference for I 2 -R ( 7), but less affinity for = 2 -AR than GBZ (14).Ag- matine, a ligand to = 2 -AR and to I 1 -R, and a potential candidate as the endogenous clonidine-displacing substance (10), had no effect on water intake.This contrasts with the potent inhibitory effect that moxonidine and clonidine, also mixed ligands to = 2 -AR and to I 1 -R, have centrally on water intake (1)(2)(3)5,12).Different from moxonidine and clonidine, agmatine has no hypotensive effect when injected centrally (15,16).It is still not known why it is hard to obtain an effect with agmatine.
IAA had no effect on water intake, suggesting that binding to I-R and not to = 2 -AR is not sufficient to inhibit water intake.This compound injected into the rostral ventrolateral medulla also causes hypotension attributed to activation of I-R (17).A possible involvement of IAA through GABA receptor binding (18,19) should have altered water intake ( 20), but such alteration did not occur in the present study.Histamine, a mixed histaminergic/imidazoline receptor ligand and a precursor of endogenous IAA (21), does not bind to = 2 -AR and also did not alter water intake.Failure to inhibit water intake by histamine can be attributed in part to its dipsogenic property expressed through activation of histaminergic receptors ( 22), but

Other behaviors
The highest dose (40 nmol) of UK 14304 produced motor deficits such as immobilization and prostration that lasted 30 min in 20% of the animals.This effect completely A B C Imidazolines, = 2 -adrenoceptors and water intake no enhancement of water intake was observed in the present study.
The selective = 2 -AR ligand mNOR (14) inhibited water intake.This is the first demonstration that a non-imidazoline selective ligand to = 2 -AR inhibits water intake.The effect of mNOR on water intake is similar in potency to the effect of the endogenous ligand noradrenaline (13) and this suggests an inhibitory role for central = 2 -AR in the control of thirst.However, neither noradrenaline (13), mNOR or GBZ are able to fully inhibit water intake by water-deprived rats.This contrasts with the near complete inhibition of water intake by clonidine (13), moxonidine (5) and UK 14304 (present study).
The results of the present study are consistent with prior evidence for an inhibitory role of = 2 -AR in the control of water intake (2,3,5).However, a straightforward interpretation of the results favoring = 2 -AR on the one hand and ruling out I-R on the other is not yet possible.Consider, first, the complete central antagonism of moxonidine by RX 821002, a selective = 2 -AR antagonist (5).Nevertheless, RX 821002 is an imidazoline molecule which can also bind to I-R (8) and moxonidine is an imidazolidine selective for I 1 -R compared to clonidine (6,14,17).Second, the inhibitory potency of the drugs on water intake seems to correlate with their molecular nature.Clonidine, moxonidine and UK 14304 are imidazolidines/imidazoline that bind to = 2 -AR and I-R and are the most potent inhibitors of water intake compared to the phenylethylamines noradrenaline, mNOR and phenylephrine (= 1 -AR agonist).To obtain the same amount of inhibition of water intake achieved with imidazolidines/ imidazoline, it is necessary to increase by four-to eight-fold all the doses below the maximum dose of noradrenaline (13) and mNOR.In addition, full inhibition of water intake is not achieved with maximum doses of noradrenaline (13), mNOR and GBZ.It is noteworthy, however, that the minimum dose needed to obtain an effect with GBZ is four times lower than the minimum dose needed to obtain an effect with mNOR.On the one hand, since GBZ binds weakly to I 1 -R, the results favor the participation of = 2 -AR.On the other hand, the higher potency of GBZ compared to mNOR suggests that binding to I-R might help.In either case, GBZ did not exert full inhibition on water intake.Thus, some kind of interaction may occur between = 2 -AR and I-R, particularly in the imidazo- line/imidazolidine domain, that is necessary for the full effect or potency of the agonists.Of course, this is not the only possible explanation and higher metabolic rates, for example, could also explain the need for higher doses of phenylethylamines to induce inhibition.Nevertheless, this hardly explains why the inhibition produced by these compounds never exceeds 70% in spite of the higher doses used.
Cumulative water intake (ml) Although evidence has pointed out distinct receptor populations (16), overlapping of = 2 -AR and I-R may also complicate the interpretation of the results.This is reflected by the ongoing controversy about the role of = 2 -AR and I-R as antihypertensive drugs (16,19,23,24).However, suggestions that an interaction between these receptors does occur are increasing (7) and our results are somehow consistent with them.Further data on receptor characterization and cloning of imidazoline-binding sites (25) will also help to clarify possible interactions with = 2 -AR.

Figure 3 .
Figure 3. Cumulative water intake by 24-h water-deprived rats after intracerebroventricular injection of: A, imidazole-4-acetic acid (IAA; 40 and 80 nmol) and B, histamine (HIS; 160 nmol), or vehicle (open squares).The number of animals per group is given in parentheses.