Investigation into the Specificity of Angiotensin II-induced Behavioral Desensitization

Angiotensin II (AngII) plays a key role in maintaining body fluid homeostasis. The physiological and behavioral effects of central AngII include increased blood pressure and fluid intake. In vitro experiments demonstrate that repeated exposure to AngII reduces the efficacy of subsequent AngII, and behavioral studies indicate that prior icv AngII administration reduces the dipsogenic response to AngII administered later. Specifically, rats given a treatment regimen of three icv injections of a large dose of AngII, each separated by 20 min, drink less water in response to a test injection of AngII than do vehicle-treated controls given the same test injection. The present studies were designed to test three potential explanations for the reduced dipsogenic potency of AngII after repeated administration. To this end, we tested for motor impairment caused by repeated injections of AngII, for a possible role of visceral distress or illness, and for differences in the pressor response to the final test injection of AngII.We found that repeated injections of AngII neither affected drinking stimulated by carbachol nor did they produce a conditioned flavor avoidance. Furthermore, we found no evidence that differences in the pressor response to the final test injection of AngII accounted for the difference in intake. In light of these findings, we are able to reject these three explanations for the observed behavioral desensitization, and, we suggest instead that the mechanism for this phenomenon may be at the level of the receptor.

Angiotensin Ii As A General Growth Factor

Angiotensin II (Ang II), a key protein in the renin-angiotensin system, can induce cardiac hypertrophy through an intracrine system as well as affect gene transcription. The receptor to Ang II responsible for this effect, AT1, has been localized to the nucleus of cell types in addition to cardiomyocytes. In this study, we induced expression of Ang II in MC3T3 osteoblasts and K7M2 osteosarcomas and measured changes in protein expression of Annexin V and matrix metalloproteinase 2 (MMP2), proteins identified previously through mass spectrometry analysis as being regulated by Ang II. Annexin V is downregulated in both immortalized murine bone (MC3T3) cells and in cancerous immortalized murine (K7M2) cells induced to express Ang II. MC3T3 cells which express Ang II show a downregulation of MMP2 expression, but Ang II-expressing K7M2 cells show an upregulation of MMP2. The differential regulation of MMP2 between the cancerous cells and noncancerous cells implicates a role for Ang in in tumor metastasis, as MMP2 is a metastatic protein. Annexin V is used as a marker for apoptosis, but nothing is known of the function of the endogenous protein. That Annexin V is potentially regulated by Ang II provides more information with which to characterize the protein and could suggest a function for Annexin V as part of a signal transduction pathway inside of the cell.

Synthesis Of Linoleate Analogs With Longer Alkyl Termini To Test For Proposed "Tail-First" Binding In SBLO-1

Lipoxygenases are nonheme-iron proteins that catalyze the oxygenation of polyunsaturated fatty acids to give conjugated diene hydroperoxides. For example, soybean lipoxygenase-1 (SBLO-1) converts linoleate into 13-(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13(S)-HPOD). Although the crystal structure of SBLO-1 has been determined, it is still unclear how the substrate binds at the active site. This absence of knowledge makes it difficult to understand the role of the enzyme during catalysis of the reaction. We hypothesize that SBLO-1 binds linoleate ¿tail-first¿, so that the methyl terminus is within a hydrophobic pocket deep within the enzyme. It is believed that the hydrophobic residue phenylalanine-557 at this site has stabilizing interactions with the terminal methyl group on linoleate. To test this hypothesis, we have developed a synthetic pathway that will yield linoleate analogs with longer fatty acid chains by 1 and 2 more carbons at the alkyl terminus. These substrates will be analyzed through kinetic assays done in combination with wild type SBLO-1 and mutants in which we have replaced phenylalanine-557 with valine.