Repeated administration of angiotensin II reduces its dipsogenic effect without affecting saline intake

Abstract

Angiotensin II (Ang II) acts at central type 1 (AT(1)) receptors to increase intake of water and saline. In vitro studies demonstrated rapid desensitization of the AT(1) receptor after Ang II exposure, and behavioural studies in rats suggest that exposure to Ang II decreases the dipsogenic potency of subsequent Ang II. Nevertheless, the effect of repeated Ang II injections on saline intake remains untested, and a reliable protocol for examining this purported behavioural desensitization has not emerged from the literature. To address these issues, we established a reliable approach to study Ang II-induced dipsetic desensitization and used this approach to test the requirement of central AT(1) receptors and the specificity of the effect for water intake. Rats given a treatment regimen of three injections of Ang II (300 ng, intracerebroventricular), each separated by 20 min, drank less water than control rats after a subsequent test injection of Ang II. The effect was relatively short lasting, dependent on the dose and timing of Ang II, and was almost completely blocked by the AT(1) receptor antagonist losartan. In further testing, when rats were given access to both water and 1.5% saline, animals that received an Ang II treatment regimen drank less water than control animals, but saline intake was unaffected. These data support previous suggestions that Ang II-induced water and saline intakes are separable. Given the role of G protein uncoupling in desensitization of the AT(1) receptor, these data are consistent with the emerging hypothesis that AT(1) receptor G protein-dependent intracellular signalling pathways are more relevant for water, but not saline, intake.

Figure 1

A treatment regimen (TR) comprising 3 injections of AngII decreased the drinking response to a subsequent test injection (T) of AngII. A timeline displaying the injection protocol is shown in panel A. Rats given a treatment regimen of AngII (300 ng) drank less water than those in the vehicle treatment regimen group after a test injection of 300 ng AngII (*p=0.043, n=5 per group; panel B) or 100 ng AngII (*p=0.021, n=7 per group; panel C).

Figure 2

The experiment illustrated in Figure 1 was repeated using new groups of rats and the total water intake over a thirty minute testing period is shown in panel A. Again, rats in the AngII treatment regimen group (300 ng AngII ×3) drank less after a test injection of AngII (100 ng) than rats in the vehicle treatment regimen group (1 μl TBS ×3; *p=0.043, n=8 per group). There were no differences in thirty minute water intake after a second test injection of AngII (100 ng) was given to all rats 24 h after the first test injection (p=0.246; panel B).

Figure 3

Injection of the AT₁-receptor antagonist losartan decreased the drinking response to AngII in a dose-dependent manner. Non-cumulative intakes by rats given various doses of losartan, administered by icv injection 20 min before 100 ng AngII, are shown in panel A (*p<0.001, †p<0.01, n=4 per group). The effects of losartan on intake were most prominent during the first 5 min of testing, when most water consumption occurred (bars with different letters differed from each other statistically, p<0.05; panel B).

Figure 4

Time-course of the antagonistic effect of losartan on AngII-induced drinking. Non- cumulative water intake by rats given icv injections of losartan 20, 40, 60, 80, or 100 min before 100 ng AngII is shown in panel A. Analysis of the data revealed a main effect of losartan on water intake (p<0.001, n=3–6 per group) and posthoc tests confirmed that the effect was statistically significant at 5 and 10 min (*p<0.001, †p<0.01). Data from the 5 min bin are shown in the histogram in panel B. Bars with different letters differed from each other statistically (p<0.05).

Figure 5

Losartan markedly attenuated the desensitizing effect of a repeated AngII treatment regimen (TR) on water intake after a test injection (T) of AngII. A timeline indicating the injection protocol is illustrated in panel A. We found a three-way interaction between a within-subject effect of time and between-subjects effects of pretreatment and treatment regimen (p=0.013, n=10–12 per group). Posthoc probes indicated that the effect of losartan occurred at the 5 min bin (*p<0.01), which is shown in the histogram in panel C. Bars with different letters differed from each other statistically (p<0.05).

Figure 6

Repeated injections of AngII affected subsequent water, but not 1.5% NaCl intake, in a two-bottle test. Rats were given a treatment regimen (TR) comprising 3 injections of AngII or vehicle, before a test injection (T) of AngII. A timeline of the experimental design is shown in panel A. Consistent with the findings above, rats given multiple injections of AngII drank less water than control rats (*p=0.017, n=9 per group; panel B). Intake of 1.5% NaCl did not differ between the groups (p=0.176; panel C). Total intake during the course of the testing period is shown in panel D (*p=0.017).