Catherine Llorens-Cortes

Expression of Angiotensin Type-1 (AT1) and Type-2 (AT2) Receptor mRNAs in the Adult Rat Brain: A Functional Neuroanatomical Review

The discovery that all components of the renin-angiotensin system (RAS) are present in the central nervous system led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurons have been visualized in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular nucleus, and supraoptic nucleus, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood-brain barrier deficient circumventricular organs are rich in angiotensin II receptors. By activating these receptors, circulating angiotensin II may act on central cardiovascular centers via angiotensinergic neurons, providing a link between peripheral and central angiotensin II systems. Among the effector peptides of the brain RAS, angiotensin II and angiotensin III have the same affinity for the two pharmacologically well-defined receptors: type 1 (AT1) and type 2 (AT2). When injected in the brain, these peptides increase blood pressure, water intake, and anterior and posterior pituitary hormone release and may modify memory and learning. The cloning of AT1 and AT2 receptor cDNAs has revealed that these receptors belong to the seven transmembrane domain receptor family. In rodents, two AT1 receptor subtypes, AT1A and AT1B, have been isolated. Using specific riboprobes for in situ hybridization histochemistry, recent studies mapped the distribution of AT1A, AT1B, and AT2 receptor mRNAs in the adult rat and found a predominant expression of AT1A and AT2 mRNA in the brain and of AT1B in the pituitary. Very limited overlap was found between the brain expression of AT1A and AT2 mRNAs. In several functional entities of the brain, such as the preoptic region, the hypothalamus, the olivocerebellary system, and the brainstem baroreflex arc, the colocalization of receptor mRNA, binding sites, and angiotensin immunoreactive nerve terminals suggests local synthesis and expression of angiotensin II receptors. In other areas, such as the bed nucleus of the stria terminalis, the median eminence, or certain parts of the nucleus of the solitary tract, angiotensin II receptors are likely of extrinsic origin. The neuronal expression of AT1A and AT2 receptors was demonstrated in the subfornical organ, the hypothalamus, and the lateral septum. By using double label in situ hybridization, AT1A receptor expression was localized in corticotropin releasing hormone but not in vasopressin containing neurons in the hypothalamus. The information is discussed together with functional data concerning the role of brain angiotensins, in an attempt to provide a better understanding of the physiological and functional roles of each receptor subtype.

Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain

Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype.

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Identification of Endocrine Cell Populations Expressing the AT1B Subtype of Angiotensin II Receptors in the Anterior Pituitary.

Angiotensin II (Ang II) participates in the regulation of anterior pituitary hormone secretion by acting either directly on the anterior pituitary or indirectly on the hypothalamus. When applied directly on pituitary cells, Ang II increases both ACTH and PRL secretion and has also been reported to affect GH secretion. Three distinct subtypes of Ang II receptors (AT1A, AT1B, and AT2) have been identified; they are unequally distributed and differently regulated in various tissues. We have previously demonstrated that only AT1A receptors are present in the hypothalamus while anterior pituitary cells express predominantly the AT1B subtype. Using in situ hybridization in combination with immunohistochemistry, the aim of the present study was to identify the phenotype of the endocrine cell expressing AT1B receptor messenger RNA (mRNA) in the anterior pituitary of adult male Sprague-Dawley rats. Expression of AT1B receptor mRNA was present in 33.9 +/- 1.0% of anterior pituitary cells. AT1B mRNA is predominantly expressed by lactotropes (78.2 +/- 2.1% of AT1B mRNA-expressing cells) and to a lower degree by corticotropes (18.3 +/- 2.1%) and is not detectable in somatotropes, mammosomatotropes, gonadotropes, or thyrotropes. These results indicate that in adult male rats, Ang II, which has been shown to be synthesized in gonadotropes, can directly stimulate PRL and ACTH release from lactotropes and corticotropes through activation of AT1B receptors. As only 53.8 +/- 2.7% of lactotropes and 23.6 +/- 2.8% of corticotropes expressed AT1B mRNA, our findings suggest a functional heterogeneity of both cell types regarding their sensitivity to Ang II.