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

Sugawara, A.M.; Miguel, T.T.; Pereira, D.T.B.; Menani, J.V.; De Luca Jr., L.A.

doi:10.1590/S0100-879X2001000900012

Abstract

Non-adrenergic ligands that bind to imidazoline receptors (I-R), a selective ligand that binds to alpha2-adrenoceptors (alpha2-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/alpha2-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 alpha2-AR agonist, alpha-methylnoradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol). The non-adrenergic 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, non-imidazoline alpha2-AR activation suppresses water intake, and suggest that the action on imidazoline sites by non-adrenergic ligands is not sufficient to inhibit water intake.

imidazolines; agmatine; UK 14304; guanabenz; alpha-methylnoradrenaline; thirst; dehydration

Braz J Med Biol Res, September 2001, Volume 34(9) 1185-1190

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

A.M. Sugawara, T.T. Miguel, D.T.B. Pereira, J.V. Menani and L.A. De Luca Jr.

Departamento de Fisiologia e Patologia, Faculdade de Odontologia, Universidade Estadual Paulista, Araraquara, SP, Brasil

References

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Abstract

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 non-adrenergic 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, non-imidazoline 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.

Key words: imidazolines, agmatine, UK 14304, guanabenz, a-methylnoradrenaline, thirst, dehydration

Introduction

Clonidine, an antihypertensive drug that binds to a₂-adrenoceptors (a₂-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 a₂-AR since it is inhibited by yohimbine, an a₂-AR antagonist (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 a₂-AR, clonidine also binds to imidazoline receptors (I-R) (4). The inhibition of water intake through activation of a₂-AR has been recently confirmed in part by the antagonism of moxonidine, a mixed I₁-R/a₂-AR ligand, by the selective a₂-AR antagonist RX 821002 (5). However, moxonidine is more selective for I₁-R than for a₂-AR (6), and RX 821002, although considered a selective a₂-AR antagonist, also has an imidazoline structure (7), thereby binding occasionally to imidazoline sites (8). This opens the possibility that imidazoline activation, in addition to a₂-AR activation, also produces inhibition of water intake. An approach to test this hypothesis 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 I-R, 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 a₂-AR, is agmatine (decarboxylated arginine) (10). Mixed I-R/a₂-AR ligands of interest (7) like guanabenz (GBZ), a guanidine, and UK 14304, an imidazoline, inhibit sodium intake (11). Imidazolidine mixed ligands to I₁-R/a₂-AR, such as clonidine and moxonidine, are known to inhibit both water and sodium intake (1-3,5,12).

The effect of a selective non-imidazoline a₂-AR ligand has not been tested on the behavior of water intake. A useful tool for this task is the a₂-AR agonist a-methylnoradrenaline (mNOR), which is likely to have the same inhibitory effect on water intake as noradrenaline (nonselective a- 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.

Material and Methods

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.

Drugs

Agmatine was purchased from Sigma, (St. Louis, MO, USA) and IAA, histamine, GBZ, UK 14304 and mNOR were purchased from RBI (Natick, MA, USA). The drugs were dissolved in 0.9% saline, but GBZ and mNOR were dissolved in acidified (pH = 4.6) 0.9% saline and UK 14304 was dissolved in 2:1 water:propyleneglycol solution.

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. Two-way (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.

Results

Injection of the selective a ₂ -AR agonist mNOR and water intake in water-deprived rats

Icv injection of mNOR (10, 20, 40, 80 and 160 nmol) reduced water intake from 39 to 76% during the whole test, except at 15 min for 10 and 40 nmol (Figure 1). Lower doses (2.5 and 5 nmol) of mNOR had no effect on water intake.

Figure 1.
Cumulative water intake by 24-h water-deprived rats after intracerebroventricular injection of a-methylnoradrenaline (mNOR; 2.5, 5, 10, 20, 40, 80 and 160 nmol) or vehicle (open squares). The number of animals per group is given in parentheses. *P<0.05

vs vehicle (Student-Newman-Keuls test).

[View larger version of this image (12 K GIF file)]

Injection of the mixed I-R/ a ₂ -AR ligands GBZ, UK 14304 and agmatine and water intake in water-deprived rats

Icv injection of GBZ reduced water intake from 30% (2.5 nmol) to 65% (5, 10, 20, 40 and 80 nmol) during the whole test (Figure 2A).

UK 14304 (5 and 10 nmol) reduced water intake by 43 and 70% from 15 to 60 min and had no effect at 120 min (Figure 2B). UK 14304 (20 and 40 nmol) reduced water intake by 90-95% from 15 to 30 min and by 60-80% from 60 to 120 min (Figure 2B).

Agmatine (80 nmol) did not alter water intake (Figure 2C).

Figure 2.
Cumulative water intake by 24-h water-deprived rats after intracerebroventricular injection of:

A, guanabenz (GBZ; 2.5, 5, 10, 20, 40 and 80 nmol); B, UK 14304 (UK; 5, 10, 20 and 40 nmol), and C, agmatine (AGM; 80 nmol) or vehicle (open squares). The number of animals per group is given in parentheses. *P<0.05 vs vehicle (Student-Newman-Keuls test).

[View larger version of this image (38 K GIF file)]

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).

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.

[View larger version of this image (30 K GIF file)]

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 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/a₂-AR ligands GBZ and UK 14304, but not agmatine, inhibited water intake by water-deprived rats. A similar effect was obtained with a-mNOR, a selective a₂-AR agonist. The non-adrenergic ligands to I-R, IAA and histamine, did not alter water intake.

GBZ is considered to be a selective a₂-AR agonist, but it also binds to I₂-R (7). UK 14304 also has preference for I₂-R (7), but less affinity for a₂-AR than GBZ (14). Agmatine, a ligand to a₂-AR and to I₁-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 a₂-AR and to I₁-R, have centrally on water intake (1-3,5,12). 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 a₂-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/I-R ligand and a precursor of endogenous IAA (21), does not bind to a₂-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 no enhancement of water intake was observed in the present study.

The selective a₂-AR ligand mNOR (14) inhibited water intake. This is the first demonstration that a non-imidazoline selective ligand to a₂-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 a₂-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 a₂-AR in the control of water intake (2,3,5). However, a straightforward interpretation of the results favoring a₂-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 a₂-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 I₁-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 a₂-AR and I-R and are the most potent inhibitors of water intake compared to the phenylethylamines noradrenaline, mNOR and phenylephrine (a₁-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 I₁-R, the results favor the participation of a₂-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 a₂-AR and I-R, particularly in the imidazoline/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.

Although evidence has pointed out distinct receptor populations (16), overlapping of a₂-AR and I-R may also complicate the interpretation of the results. This is reflected by the ongoing controversy about the role of a₂-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 a₂-AR.

Acknowledgments

The authors thank S.P. Barbosa, R.C. Queiróz, S. Foglia and A. Vítor for technical assistance, and S.D. Malavolta for secretarial assistance.

Address for correspondence: L.A. De Luca Jr., Departamento de Fisiologia e Patologia, Faculdade de Odontologia, UNESP, Rua Humaitá, 1680, 14801-903 Araraquara, SP, Brasil. Fax: +55-16-201-6488 or 222-4823. E-mail: lucajr@foar.unesp.br

Research supported by CNPq-PRONEX and FAPESP. A.M. Sugawara was the recipient of a graduate fellowship from FAPESP. T.T. Miguel and D.T.B. Pereira were recipients of undergraduate fellowships from FAPESP and CNPq, respectively. Part of a Master's thesis presented by A.M. Sugawara to the Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brazil. Received April 13, 2000. Accepted June 5, 2001.

1. De Luca Jr LA & Menani JV (1997). Multifactorial control of water and saline intake: role of a₂-adrenoceptors. Brazilian Journal of Medical and Biological Research, 30: 497-502.
2. Fregy MJ, Rowland NE & Greenleaf JE (1984). A role for presynaptic a₂-adrenoceptors in angiotensin II-induced drinking in rats. Brain Research Bulletin, 12: 393-398.
3. Ferrari AC, Camargo LAA, Saad WA, Renzi A, De Luca Jr LA & Menani JV (1990). Clonidine and phenylephrine injected into the lateral hypothalamus inhibit water intake in rats. Brain Research, 522: 125-130.
4. Brown CM, MacKinnon AC, McGrath JC, Spedding M & Kilpatrick AT (1990). a₂-Adrenoceptor subtypes and imidazoline-like binding sites in the rat brain. British Journal of Pharmacology, 99: 803-809.
5. Menani JV, Sato MA, Haikel L, Vieira AA, de Andrade CAF, da Silva DCF, Renzi A & De Luca Jr LA (1999). Central moxonidine on water and NaCl intake. Brain Research Bulletin, 49: 273-279.
6. Ernsberger P, Friedman JE & Koletsky RJ (1997). The I₁-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease. Journal of Hypertension, 15 (Suppl 1): S9-S23.
7. Hieble JP & Ruffolo Jr RR (1995). Possible structural and functional relationships between imidazoline receptors and a₂-adrenoceptors. Annals of the New York Academy of Sciences, 763: 8-21.
8. Callado LF, Gabilondo AM & Meana JJ (1996). [³H]RX821002 (2-methoxyidazoxan) binds to a₂-adrenoceptor subtypes and a non-adrenoceptor imidazoline binding site in rat kidney. European Journal of Pharmacology, 316: 359-368.
9. Lehman J, Koenig-Bérard E & Vitou P (1989). The imidazoline-preferring receptor. Life Sciences, 45: 1609-1615.
10. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R & Reis DJ (1994). Agmatine: an endogenous clonidine-displacing substance in the brain. Science, 263: 966-968.
11. Sugawara AM, Miguel TT, de Oliveira LB, Menani JV & De Luca Jr LA (1999). Noradrenaline and mixed a₂-adrenoceptor/imidazoline-receptor ligands: effects on sodium intake. Brain Research, 839: 227-234.
12. Yada MM, de Paula PM, Menani JV, Renzi A, Camargo LAA, Saad WA & De Luca Jr LA (1997). Receptor-mediated effects of clonidine on need-induced 3% NaCl and water intake. Brain Research Bulletin, 42: 205-209.
13. Yada MM, de Paula PM, Menani JV & De Luca Jr LA (1997). Central a-adrenergic agonists and need-induced 3% NaCl and water intake. Pharmacology, Biochemistry and Behavior, 57: 137-143.
14. Ernsberger P, Graves ME, Graff LM, Zakieh N, Nguyen P, Collins LA, Westbrooks KL & Johnson GG (1995). I₁-Imidazoline receptors: definition, characterization, distribution, and transmembrane signaling. Annals of the New York Academy of Sciences, 763: 22-42.
15. Head GA (1995). Importance of imidazoline receptors in the cardiovascular actions of centrally acting antihypertensive agents. Annals of the New York Academy of Sciences, 763: 531-540.
16. Reis DJ & Piletz JE (1997). The imidazoline receptor in control of blood pressure by clonidine and allied drugs. American Journal of Physiology, 273: R1569-R1571.
17. Ernsberger PR, Westbrooks KL, Christen MO & Schäfer SG (1992). A second generation of centrally acting antihypertensive agents act on putative I₁-imidazoline receptors. Journal of Cardiovascular Pharmacology, 20 (Suppl 4): S1-S10.
18. Bormann J & Feigenspan A (1995). GABAc receptors. Trends in Neurosciences, 18: 515-519.
19. Guyenet PG (1997). Is the hypotensive effect of clonidine and related drugs due to imidazoline binding sites? American Journal of Physiology, 273: R1580-R1584.
20. Jones DL & Mogenson GJ (1982). Central injections of spiperone and GABA: attenuation of angiotensin II stimulated thirst. Canadian Journal of Physiology and Pharmacology, 60: 720-726.
21. Green JP (1989). Histamine and serotonin. In: Siegel GJ, Agranoff BW, Albers RW & Molinoff PB (Editors), Basic Neurochemistry Raven Press, New York.
22. Kraly FS (1990). Drinking elicited by eating. Progress in Psychobiology and Physiological Psychology, 14: 67-131.
23. Ernsberger P & Haxhiu MA (1997). The I₁-imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla. American Journal of Physiology, 273: R1572-R1579.
24. Eglen RM, Hudson AL, Kendall DA, Nutt DJ, Morgan NG, Wilson VG & Dillon MP (1998). "Seeing through a glass darkly": casting light on imidazoline 'I' sites. Trends in Pharmacological Sciences, 19: 381-390.
25. Piletz JE, Ivanov TR, Sharp JD, Ernsberger P, Chang CH, Pickard RT, Gold G, Roth B, Zhu H, Jones JC, Baldwin J & Reis DJ (2000). Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization. DNA and Cell Biology, 19: 319-329.

Figure 1. Cumulative water intake by 24-h water-deprived rats after intracerebroventricular injection of a-methylnoradrenaline (mNOR; 2.5, 5, 10, 20, 40, 80 and 160 nmol) or vehicle (open squares). The number of animals per group is given in parentheses. *P<0.05 vs vehicle (Student-Newman-Keuls test).

Figure 2. Cumulative water intake by 24-h water-deprived rats after intracerebroventricular injection of: A, guanabenz (GBZ; 2.5, 5, 10, 20, 40 and 80 nmol); B, UK 14304 (UK; 5, 10, 20 and 40 nmol), and C, agmatine (AGM; 80 nmol) or vehicle (open squares). The number of animals per group is given in parentheses. *P<0.05 vs vehicle (Student-Newman-Keuls test).

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.

Correspondence and Footnotes

Publication Dates

Publication in this collection
16 Aug 2001
Date of issue
Sept 2001

History

Received
13 Apr 2000
Accepted
05 June 2001

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. De Luca Jr LA & Menani JV (1997). Multifactorial control of water and saline intake: role of a₂-adrenoceptors. Brazilian Journal of Medical and Biological Research, 30: 497-502.

[2] 2. Fregy MJ, Rowland NE & Greenleaf JE (1984). A role for presynaptic a₂-adrenoceptors in angiotensin II-induced drinking in rats. Brain Research Bulletin, 12: 393-398.

[3] 3. Ferrari AC, Camargo LAA, Saad WA, Renzi A, De Luca Jr LA & Menani JV (1990). Clonidine and phenylephrine injected into the lateral hypothalamus inhibit water intake in rats. Brain Research, 522: 125-130.

[4] 4. Brown CM, MacKinnon AC, McGrath JC, Spedding M & Kilpatrick AT (1990). a₂-Adrenoceptor subtypes and imidazoline-like binding sites in the rat brain. British Journal of Pharmacology, 99: 803-809.

[5] 5. Menani JV, Sato MA, Haikel L, Vieira AA, de Andrade CAF, da Silva DCF, Renzi A & De Luca Jr LA (1999). Central moxonidine on water and NaCl intake. Brain Research Bulletin, 49: 273-279.

[6] 6. Ernsberger P, Friedman JE & Koletsky RJ (1997). The I₁-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease. Journal of Hypertension, 15 (Suppl 1): S9-S23.

[7] 7. Hieble JP & Ruffolo Jr RR (1995). Possible structural and functional relationships between imidazoline receptors and a₂-adrenoceptors. Annals of the New York Academy of Sciences, 763: 8-21.

[8] 8. Callado LF, Gabilondo AM & Meana JJ (1996). [³H]RX821002 (2-methoxyidazoxan) binds to a₂-adrenoceptor subtypes and a non-adrenoceptor imidazoline binding site in rat kidney. European Journal of Pharmacology, 316: 359-368.

[9] 9. Lehman J, Koenig-Bérard E & Vitou P (1989). The imidazoline-preferring receptor. Life Sciences, 45: 1609-1615.

[10] 10. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R & Reis DJ (1994). Agmatine: an endogenous clonidine-displacing substance in the brain. Science, 263: 966-968.

[11] 11. Sugawara AM, Miguel TT, de Oliveira LB, Menani JV & De Luca Jr LA (1999). Noradrenaline and mixed a₂-adrenoceptor/imidazoline-receptor ligands: effects on sodium intake. Brain Research, 839: 227-234.

[12] 12. Yada MM, de Paula PM, Menani JV, Renzi A, Camargo LAA, Saad WA & De Luca Jr LA (1997). Receptor-mediated effects of clonidine on need-induced 3% NaCl and water intake. Brain Research Bulletin, 42: 205-209.

[13] 13. Yada MM, de Paula PM, Menani JV & De Luca Jr LA (1997). Central a-adrenergic agonists and need-induced 3% NaCl and water intake. Pharmacology, Biochemistry and Behavior, 57: 137-143.

[14] 14. Ernsberger P, Graves ME, Graff LM, Zakieh N, Nguyen P, Collins LA, Westbrooks KL & Johnson GG (1995). I₁-Imidazoline receptors: definition, characterization, distribution, and transmembrane signaling. Annals of the New York Academy of Sciences, 763: 22-42.

[15] 15. Head GA (1995). Importance of imidazoline receptors in the cardiovascular actions of centrally acting antihypertensive agents. Annals of the New York Academy of Sciences, 763: 531-540.

[16] 16. Reis DJ & Piletz JE (1997). The imidazoline receptor in control of blood pressure by clonidine and allied drugs. American Journal of Physiology, 273: R1569-R1571.

[17] 17. Ernsberger PR, Westbrooks KL, Christen MO & Schäfer SG (1992). A second generation of centrally acting antihypertensive agents act on putative I₁-imidazoline receptors. Journal of Cardiovascular Pharmacology, 20 (Suppl 4): S1-S10.

[18] 18. Bormann J & Feigenspan A (1995). GABAc receptors. Trends in Neurosciences, 18: 515-519.

[19] 19. Guyenet PG (1997). Is the hypotensive effect of clonidine and related drugs due to imidazoline binding sites? American Journal of Physiology, 273: R1580-R1584.

[20] 20. Jones DL & Mogenson GJ (1982). Central injections of spiperone and GABA: attenuation of angiotensin II stimulated thirst. Canadian Journal of Physiology and Pharmacology, 60: 720-726.

[21] 21. Green JP (1989). Histamine and serotonin. In: Siegel GJ, Agranoff BW, Albers RW & Molinoff PB (Editors), Basic Neurochemistry Raven Press, New York.

[22] 22. Kraly FS (1990). Drinking elicited by eating. Progress in Psychobiology and Physiological Psychology, 14: 67-131.

[23] 23. Ernsberger P & Haxhiu MA (1997). The I₁-imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla. American Journal of Physiology, 273: R1572-R1579.

[24] 24. Eglen RM, Hudson AL, Kendall DA, Nutt DJ, Morgan NG, Wilson VG & Dillon MP (1998). "Seeing through a glass darkly": casting light on imidazoline 'I' sites. Trends in Pharmacological Sciences, 19: 381-390.

[25] 25. Piletz JE, Ivanov TR, Sharp JD, Ernsberger P, Chang CH, Pickard RT, Gold G, Roth B, Zhu H, Jones JC, Baldwin J & Reis DJ (2000). Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization. DNA and Cell Biology, 19: 319-329.