Kevin L. Grove

Angiotensin II receptor subtypes in the rat brain.

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1,Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 greater than 10(-4) M). DuP 753 sensitive binding sites (designated AII alpha subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AII beta) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.

Discrimination of angiotensin II receptor subtype distribution in the rat brain using non-peptidic receptor antagonists

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AII alpha subtype) or PD123177 (designated AII beta subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AII alpha and AII beta subtypes.

Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain.

Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1,isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1,isoleucine8 angiotensin II binding was strongly inhibited by 30 mM beta-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AII alpha) 125I-sarcosine1,isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AII beta) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AII alpha) receptor subtype.

The AT2 angiotensin receptor subtype predominates in the 18 day gestation fetal rat brain

The angiotensin II receptor subtype-specific antagonists Dup 753 (AT1) and PD 123177 (AT2) were used to characterize the angiotensin II receptor subtypes present in 18 day gestation fetal Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rat brain using in vitro receptor autoradiography. The AT2 subtype was predominant in the brain of both rat strains, even in areas that display predominantly the AT1 subtype in the adult rat brain.

Angiotensin II and non-angiotensin II displaceable binding sites for [3H]losartan in the rat liver

By virtue of the more than 1000-fold selectivity of losartan (DuP 753) for the AT1 angiotensin II (AII) receptor subtype compared with the AT2 subtype, [3H]losartan may be a useful radioligand for studies of the AT1 receptor subtype. Comparison of Bmax values in the liver obtained from saturation isotherms using [3H]losartan (Bmax = 194 pmol/g tissue) and [125I]sarcosine1,isoleucine8 angiotensin II (Bmax = 20 pmol/g tissue) indicated that the AII receptor concentration was approximately 10% that of the [3H]losartan binding sites. In addition, AII at concentrations as high as 10 microM displaced less than one-third of specific [3H]losartan binding in the liver and less than 80% in the whole adrenal. The presence of non-AII displaceable [3H]losartan binding in the liver did not appear to result from metabolism of the radioligand since HPLC analysis of free and bound 3H revealed that greater than 90% of the 3H eluted at the same time as the parent [3H]losartan. This suggests that [3H]losartan binds with high affinity to a site(s) other than angiotensin II receptors in the rat liver.

Angiotensin II Receptors in the Ventral Portion of the Bed Nucleus of the Stria terminalis

In vitro receptor autoradiography, using the radiolabeled angiotensin II (Ang II) antagonist 125I-sar1,ile8 Ang II (125I-SI Ang II; 250 pM) in the absence or presence of 1 microM Ang II, was used to identify Ang II receptor binding sites in the preoptic-anterior hypothalamic (POAH) brain region of cycling female rats. A nucleus within this region, lateral to the organum vasculosum of the lamina terminalis and ventral to the anterior commissure, displayed a discrete locus of 125I-SI Ang II binding sites (385 fmol/g tissue). This nucleus, which corresponds to the area of the POAH from which Ang II is most effective at eliciting luteinizing hormone release, has been identified as the ventral portion of the bed nucleus of the stria terminalis (BSTV) by the rat brain atlas of Paxinos and Watson. The selective nonpeptidic Ang II alpha receptor antagonist Dup 753 completely inhibited the binding of 125I-SI Ang II to the BSTV and other hypothalamic nuclei, suggesting that these receptors are of the Ang II alpha subtype.

Angiotensin II and the locus coeruleus

The locus coeruleus (LC) is a putative site of action for angiotensin II in the brain. Immunocytochemical studies have identified angiotensin II-like immunoreactive material in nerve terminals innervating the LC, and the LC contains one of the highest densities of angiotensin II receptor binding sites in the rat brain. Recent studies using selective neurotoxins suggest that the binding sites for angiotensin II in the LC are present on noradrenergic perikarya. Angiotensin II receptors are now known to exist as two subtypes that are distinguishable both pharmacologically and biochemically. Radioligand binding studies using agonists and antagonists selective for these angiotensin II receptor subtypes indicate that the rat LC contains a mixture of the two known angiotensin II receptor subtypes, but that the PD123177-sensitive AII beta receptor subtype is predominant. Comparisons of spontaneously hypertensive rats with normotensive rats indicates that angiotensin II and its receptors in the LC are elevated in the hypertensive rat strain. Studies of the biochemical and physiological actions of angiotensin II in the LC have not yet established an agreed-upon function for angiotensin II in this nucleus.

Angiotensin II receptor binding sites in the ventral portion of the bed nucleus of the stria terminalis are reduced by interruption of the medial forebrain bundle

Many techniques have been utilized to discern the localization of angiotensin II (Ang II) receptors to specific cellular components (glia, neuronal cell bodies and nerve terminals) in the brain. In the present study, we used lesioning techniques to localize Ang II receptors to cellular components in the rat forebrain. In the first experiment, axons ascending to the hypothalamus and forebrain from neurons in the brainstem were destroyed by unilaterally cutting the medial forebrain bundle (MFB). In the second experiment, a single injection of the neurotoxin, ibotenic acid, was injected unilaterally into the ventral portion of the bed nucleus of the stria terminalis (BSTV) to destroy neuronal cell bodies, thus determining if Ang II receptors are present on neuronal cell bodies. In both experiments, the animals were sacrificed after two weeks recovery and the brains processed for in vitro receptor autoradiography using 125I-sar1,ile8 Ang II (125I-SI Ang II). Unilateral knife-cut lesions of the MFB caused a significant reduction in 125I-SI Ang II binding in the BSTV (30+/-6%) and the piriform cortex (PC; 26+/-4%) ipsilateral to the knife cut. Unilateral injection of the neurotoxin into the BSTV failed to alter 125I-SI Ang II binding in this nucleus. These experiments suggest that at least a subpopulation of Ang II receptors in the BSTV and PC are located on terminals of neurons that have their cell bodies in the brainstem and their axons in the MFB.

Pertussis toxin blocks the dipsogenic actions of carbachol, but does not block the dipsogenic and pressor actions of angiotensin II.

Rats were tested for dipsogenic and pressor responses to intracerebroventricularly (icv) administered Ang II and for dipsogenic responses to icv administered carbachol in the absence and presence of pertussis toxin, also administered icv. Pertussis toxin did not inhibit the pressor or dipsogenic responses to Ang II, but did inhibit the dipsogenic responses to carbachol. This suggests that the pressor and dipsogenic responses to Ang II in the brain are not mediated by a pertussis toxin-sensitive G protein, but that the muscarinic cholinergic dipsogenic response is mediated by a pertussis toxin-sensitive G protein.

Differences between perinatal angiotensin binding in the brains of SHR and WKY rats

A growing body of evidence suggests that angiotensin may have a functional role in growth and development, in addition to its classical role in the maintenance of body water homeostasis. Components of the renin-angiotensin system have been identified in the rat fetus. Because of the association between the renin-angiotensin system and hypertension, we quantified angiotensin receptor binding sites in the brains of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats during perinatal development. Using in vitro receptor autoradiography we identified specific 125I-Sar1,Ile8 AII binding in several areas of the brains of perinatal rats of both strains and observed significant differences in the concentration of binding sites, at different ages in several brain nuclei. With the knowledge that components of the renin-angiotensin system appear early in development and are known to have an association with cellular growth, it is possible that an irregularity in this system occurring during neurogenesis could contribute to developmental abnormalities, as well as subsequent hypertension.