Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II

Abstract

Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

Figure 1

Illustration of the approximate boundaries of the microdissected tissue used to measure MAP kinase. One hundred micron tissue sections from the anterior (A) and posterior (D) lamina terminalis were dissected to include AngII-responsive structures in the ventral and the dorsal lamina terminalis, respectively. Atlas plates were modified from Swanson (2003). Abbreviations not used elsewhere: ac, anterior commissure; fx, fornix; LPO, lateral preoptic area; LV, lateral ventricle; MPO, medial preoptic nucleus; oc, optic chiasm; Pe, periventricular hypothalamus; 3V, third ventricle; ventral hippocampal commissure (vhc).

Figure 2

MAP kinase activity in tissue containing the ventral or the dorsal portion of the lamina terminalis after exposure to a treatment regimen of AngII, SII, or vehicle. Rats were given repeated injections of AngII (3 × 300 ng in 1 μl TBS), SII (3 × 30 μg in 1 μl TBS), or vehicle (3 × 1 μl TBS) before all rats received a test injection of AngII (100 ng in 1 μl TBS). Brains were removed and tissue containing either the ventral or the dorsal portion of the lamina terminalis was homogenized and analyzed by SDS-PAGE followed by Western blotting for phosphorylated and total p44/42 MAP kinase (ERK1/2). Representative Western blots of phosphorylated and total MAP kinase are shown in panels A and B and in D and E for anterior and posterior tissue, respectively. The approximate location of the two closest molecular weight markers is shown to the left of each image for phosphorylated and total MAP kinase. Optical density (OD) was measured and quantification of activated MAP kinase, expressed as the percent of total MAP kinase, is shown for the anterior (C) and posterior (F) samples. No group differences were detected in either region (p-values>0.05; n=9 per group).

Figure 3

Pretreatment with the MAP kinase inhibitor, U0126, prevented the behavioral desensitization caused by repeated injections of AngII. A) Timeline of the experiment. B) There was a significant Time × Pretreatment × Treatment Regimen interaction and posthoc tests found that the effect was driven by differences in intake 5 min after the AngII test injection (*p<0.05; n=5-7 per group). As shown in panel C, pretreatment with U0126 prevented the effect of an AngII treatment regimen on 5-min water intake (p<0.05). Bars with different letters were significantly different. D) Thirty minute intake by rats given pretreatment with U0126 and repeated injections of AngII was greater than that by rats given repeated injections of AngII without U0126 (*p<0.05 U0126/AngII vs Veh/AngII).

Figure 4

G protein activation is not required for AngII-induced behavioral desensitization. A) Rats given an AngII or an SII treatment regimen had reduced 30 min intake (p<0.05; n=17 per group). B) A significant difference in non-cumulative intake was observed at 5 min and 10 min after the test injection (*p<0.05). C) Rats given an AngII or an SII treatment regimen drank less than vehicle-treated rats at 5 and 10 min after the test injection (p<0.05). Rats treated with SII also drank less than AngII-treated rats in the 5 and 10 min time bins (p<0.05). D) SII caused a reduction in AngII-stimulated intake only when given immediately before AngII injection (p<0.05; n=7-8 per group). Bars with different letters are different from each other.