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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 29, 2001 as doi:10.1096/fj.00-0797fje. |
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,
Institute of Molecular Pharmacology, Berlin, Germany;
* Genome Research,
Hypertension Research, Max-Delbrueck Center for Molecular Medicine, Berlin-Buch, Germany;
Institute of Pharmacology and Toxicology, University of Magdeburg, Magdeburg, Germany; and
Department of Cardiology and Pneumology, University Hospital, Benjamin Franklin Free University of Berlin, Berlin, Germany
2Correspondence: Institute of Molecular Pharmacology, Robert-Roessle-Str. 10, 13125 Berlin, Germany. E-mail maul{at}fmp-berlin.de
SPECIFIC AIMS
We addressed the hypothesis that angiotensin II (AII) is involved in alcohol-consuming behavior and investigated the alcohol intake of mice harboring a rat angiotensinogen transgene and mice lacking the angiotensinogen gene in a two-bottle choice paradigm. The influence of an inhibitor of the angiotensin-converting enzyme (ACE) and a dopamine receptor antagonist was studied to further explain the effects in the transgenic mice.
PRINCIPAL FINDINGS
1. Alcohol consumption is directly correlated with angiotensin
levels
To clarify the role of AII in alcohol consumption, we tested the
correlation between endogenous AII levels and voluntary alcohol intake
in transgenic mice expressing a rat angiotensinogen transgene (TGM123),
knockout mice lacking the angiotensinogen gene (TLM), and their
respective controls. TGM123 mice (n=10) and their wild-type
littermates (n=9) were held in a free choice paradigm for 7
wk. They were offered a choice between water and 10% (v/v) ethanol.
This concentration of alcohol has been shown to provoke aversive
responses in mice. Indeed, the mice avoided drinking from the alcohol
bottle, so that the alcohol preference ratio was smaller than 0.5.
Nonetheless, ratio (Fig. 1A
) as well as alcohol consumption (Fig. 1B
) were
significantly increased by at least a third in TGM123 mice compared to
their wild-type littermates.
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Conversely, TLM mice (n=12) showed a smaller alcohol preference ratio than their wild-type littermates (n=12) when being held in a free choice paradigm for 4 wk. Since mutant mice suffered from severe disturbances in fluid homeostasis due to defects in their renal glomeruli, the total fluid consumption of TLM mice was about twice that of controls. Nevertheless, even these mice drank substantially less alcohol than the controls.
2. The difference in alcohol consumption is unlikely to be calorie
driven or due to differences in taste aversion
To see whether TGM123 mice would exhibit a higher affinity to any
fluid other than water, we tested them for their intake of a sweet and
a bitter tastant in a free choice paradigm. In one experiment, TGM123
mice (n=10) and their wild-type littermates (n=9)
were given the choice between tap water and 1.7% (w/v) sucrose in tap
water over a period of 6 days. TGM123 mice were not different from
controls in their preference for the sucrose solution (Fig. 1C
). Therefore, the increased alcohol consumption in TGM123
mice is unlikely to be calorie driven. We also detected no differences
between TGM123 mice (n=11) and their wild-type littermates
(n=10) in their aversion to bitter beverages when the
animals were offered increasing concentrations of a quinine solution
vs. tap water (Fig. 1D
).
3. Inhibition of ACE and blockade of dopamine receptors blunt the
transgenic effect on alcohol intake
Spirapril, a lipophilic nonpeptidic ACE inhibitory drug, was used
to test whether impaired synthesis of AII would blunt the transgenic
effect. An interesting feature of spirapril is its central
availability. In fact, brain membrane preparations of treated mice
administered with 10 mg per kg body weight spirapril via a feeding
needle showed a 40.2% reduction in ACE activity; this effect was
specific as the inhibition could be relieved by several washing steps.
After they had been held in the free choice paradigm for 4 wk, TGM123 mice (n=13) and TLM mice (n=12) were given spirapril. Indeed, the substance significantly reduced the elevated preference ratio in TGM123 from 176% to 141% of the mean value in controls. As expected, spirapril did not reduce the alcohol consumption in TLM mice.
Assuming that dopaminergic systems might be involved in the
AII-dependent elevation of voluntary alcohol consumption, animals being
held in a free choice paradigm were administered with 1 mg per kg body
weight fluphenazine via the consumed tap water as calculated from their
water consumption. The consumption of alcohol in TGM123 mice was
significantly reduced upon administration of the drug to reach the
alcohol intake levels observed in the wild-type littermates (Fig. 1B
).
4. Introduction of the angiotensinogen transgene increases
sensitivity to central alcohol effects
Experiments were performed to address additional indices of
central alcohol action, because changes in alcohol intake may not
necessarily be related to the specific pharmacological effects of
alcohol. TGM123 mice showed a stronger reduction of their horizontal
activity in an activity meter over a 10 min test period after
application of 2.5 g ethanol per kg body weight (Fig. 2A
). Moreover, it took TGM123 mice significantly more time to reestablish
their righting reflex after administration of 4 g ethanol per kg
body weight (Fig. 2B
). These differences in sensitivity to
alcohol effects as well as those in alcohol consumption are not due to
unequal alcohol clearing rates, as plasma ethanol concentrations were
not different at two time points after injecting the animals with
alcohol.
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CONCLUSIONS
Although potential involvement of the renin-angiotensin system in alcohol-consuming behavior in animals has long been suggested, the function of AII in this context remained unclear. We have demonstrated a direct correlation between endogenous AII levels and voluntary alcohol intake in genetically altered mice. Alcohol consumption in TGM123 mice, which show an elevation of AII levels, was significantly increased. Conversely, the alcohol preference ratio in TLM mice lacking the angiotensinogen gene was smaller than in their controls. Despite their excessive total fluid consumption, the TLM mice drank even substantially less alcohol than the controls. Because TLM mice otherwise display completely normal activity in all behavioral paradigms tested, we assume that they selectively avoid ingesting the alcoholic beverage in an attempt to compensate for the fluid loss caused by glomeruli dysfunction.
Our results are supported by the finding that i.c.v. infusion of AII increased alcohol intake in rats. However, other authors reported a reduction of alcohol intake in rats induced by exogenously administered AII. These latter experiments, however, used a different mode of administration of the alcoholic beverage: a limited access procedure. We tested alcohol consumption on animals that had the drug freely available 24 h a day. Moreover, we measured alcohol consumption without exposing the animals to any invasive application procedures. This may provide improved validity of the behavioral parameters investigated. The most likely explanation for the differences is that we and the authors of studies with similar findings analyzed animals in which AII levels are changed in the brain. The other authors, however, modified only the systemic levels of the peptide, which is unable to cross the blood–brain barrier. They suggested that systemic AII might act as a short-term satiety signal for alcohol, leading to a reduction in alcohol intake. This would be difficult to reconcile in view of the reduction in alcohol consumption upon the suppression of the AII synthesis by ACE inhibitors. However, since most of these drugs cross the blood–brain barrier, they may influence alcohol consumption mainly by decreasing central AII levels, thus eliciting the same effect on alcohol drinking as observed in TLM mice. Indeed, the ACE inhibitor spirapril, which crossed the blood–brain barrier, suppressed the transgene effect in our experiments. However, the effect of spirapril on ACE did not completely compensate for the AII-induced increase in alcohol consumption. This may certainly be attributed to the incomplete inhibition of the enzyme activity. Even in the case of a total ACE inhibition, however, alternative pathways for AII synthesis may be unaffected.
Brain AII is a part of the systems that regulate thirst and sodium intake. This could raise the concern that changes in alcohol intake may not be related to the specific pharmacological effects of alcohol. However, TGM123 mice showed a stronger alcohol-associated reduction in their horizontal activity and prolonged sedation at a high alcohol dose. Thus, in these tests TGM123 mice exhibited a higher sensitivity to alcohol. On the other hand, they consumed larger amounts of alcohol in the free choice paradigm. It is interesting to note that mice lacking the D2 receptor gene were less sensitive to alcohol-induced ataxia than their wild-type littermates whereas they ingested lower amounts of alcohol in free choice experiments. It is known that AII stimulates dopamine release in the brain, and angiotensin receptors are abundantly expressed in brain areas such as the nucleus accumbens, where dopaminergic transmission has been strongly implicated in alcohol self-administration and sensitivity. We found that the voluntary consumption of alcohol of TGM123 mice was significantly reduced upon administration of the dopamine receptor antagonist fluphenazine. Thus, increased alcohol intake in angiotensin-overexpressing mice may relate to an interaction of AII with dopaminergic systems.
Taken together, our results shed new light on the connection between AII and alcohol-consuming behavior. They provide a further contribution to the dissection of the genetic architecture of a devastating disease. Recently, a connection was shown between the ACE DD genotype associated with high ACE activity in humans and an increased susceptibility to alcoholism. The elucidation of the mechanism of AII action in regulating alcohol drinking behavior may therefore reveal new pharmacological paradigms for treating alcohol abuse.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0797fje ; to cite this
article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0797fje 
This article has been cited by other articles:
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