A Cre-loxP solution for defining the brain renin-angiotensin system. Focus on "Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain".

UNLOCKING THE MYSTERY of the brain renin-angiotensin system (RAS) and its role in cardiovascular physiology has been a theme for state-of-the-art research for 30 years. Each time a new, more precise tool becomes available, the problem is revisited. In this release of Physiological Genomics, Sinnayah et al. (24) present a report on a new way to analyze the role of specific genes in specific cardiovascular circuits in the mouse brain using Cre recombinase delivered by viral vectors. An independent RAS was first proposed to exist in the brain based on radioimmunoassay and enzyme assays (7). The concept was controversial because the biochemistry of the circulating RAS had been well worked out by 1970. The whole cascade was known. Renin from the kidney was the ratelimiting enzyme to specifically hydrolyze angiotensinogen (AGT) in the liver to angiotensin I (ANG I). ANG I, a decapeptide, was converted by angiotensin converting enzyme (ACE) to the octapeptide ANG II. ANG II was the active component. End of story. Ganten et al. in 1971 (7) found evidence of angiotensin-forming enzyme in brain tissue, which was presumably renin. About that time ANG II had become very interesting, physiologically and behaviorally. When ANG II is injected into the brain it elicits a pressor response and, if water is available, a drinking response. However, in both cases there was controversy about whether the effects reflected the actions of the circulating RAS or required a postulated brain RAS. After all, circulating RAS increased blood pressure, and some of the most efficacious treatments for high blood pressure today are oral or systemically delivered anti-ANG II drugs. Drinking behavior obviously requires neural circuits in the brain. It was reasoned that the effects in the brain were isolated from circulating RAS by the blood-brain barrier (BBB). However, there are small areas of the brain which have no BBB, the circumventricular organs (CVOs). The role of these sites was hotly contended (2). With this background, there has always been a high level of proof demanded for the brain RAS. One of the techniques applied was iontophoresis, which showed that ANG II activated neurons in the brain and in the subfornical organ (SFO) (18). A peptide antagonist of ANG II, saralasin (which was new at the time), inhibited those neurons, suggesting ANG II was a neurotransmitter. Simply injecting saralasin alone into the brain of hypertensive rats (SHR) temporarily reduced high blood pressure (19). This occurred even in the absence of renin when the SHR rats were bilaterally nephrectomized (19). Immunocytochemistry with ANG II antibodies showed a clear distribution of ANG II-like staining in the hypothalamus and CVOs such as the SFO, organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus (MnPO), and the median eminence. The criticism of those results was that the ANG II antibodies used were polyclonal and therefore not definitive. What was needed was actual measurement of the ANG II protein. This was the achieved with high-performance liquid chromatography (6, 20). Brain levels of ANG II were higher than circulating levels of ANG II. Having now shown that ANG II was in the brain, the next level of proof required not only measuring the protein but demonstrating that it had been synthesized in the brain. To the rescue came what was then a novel technique, Northern blotting, which showed mRNA for AGT in the brain (3). Renin mRNA was also claimed (5). But blots do not show where in the brain the mRNA is located or expressed. Stornetta et al. (26) identified the site of synthesis of AGT in mRNA using in situ hybridization in glial cells in the hypothalamus. Another new technique at that time was about to shed much more light on the brain RAS. Mendelsohn et al. (12), using autoradiography with pseudocolor imaging, were able to show maps of the distribution ANG II receptor binding sites located in many nuclei of the brain in addition to the CVOs. This proved that there were sites within the brain that had evolved to be activated by ANG II in the brain. Minute injections of ANG II into key brain nuclei showed that ANG II could differentially produce a pressor effect, elicit drinking, release vasopressin, inhibit the baroreflex and increase sympathetic nervous system activity (11, 22). The realization that there was a separate brain RAS, independent of a circulating RAS, gave rise to the more general concept of local or tissue angiotensins. An even bigger breakthrough came when Whitebread et al. (27) and Chiu et al. (4) showed that there were two subtypes of the ANG II receptor, AT1 and AT2. From this has sprung not only insight into genes involved in brain function, but also the highly successful angiotensin receptor blockade (ARB) class of antihypertensive drugs. Cloning the angiotensin receptors has led to insight into signaling pathways and genetic manipulation. With the development of transgenic mice (17), the first transgenic mouse relevant to RAS was one that demonstrated AGT overexpression by a metallothionein promoter (16). This was followed by the transgenic mouse with an AGT overexpression in brain and liver which had high blood pressure (10). But what about renin? Renin exists in only one form in humans, Ren-1, but in two forms in mice, Ren-1 and Ren-2. The data showing renin in the brain with Northern blots were weak (some critics say ghostly). By inserting a mouse Ren-2 gene into a transgenic rat [TGR(mREN-2)27], Mullins et al. (15) reported fulminant hypertension in transgenic rats. These rats developed high Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org). Address for reprint requests and other correspondence: M. Ian Phillips, Univ. of South Florida, 4202 E. Fowler Ave., ADM200, Tampa, FL 33620 (E-mail: [email protected]). Physiol Genomics 18: 1–3, 2004; 10.1152/physiolgenomics.00115.2004.