Mapping of angiotensin AT1 receptors in the rat limbic system

The AT1 receptor is one of the two receptor subtypes able to bind angiotensin II. In the present study, immunohistochemical examination of the distribution of the AT1 receptor in several limbic structures of female rats has been done, revealing new aspects of the distribution of AT1-positive cells. The presence of AT1 receptor expressing cells in the hippocampus and the amygdala is described, but their distribution in these regions has not been examined in a detailed way. We found some notable differences in the distribution of these cells: in female rats, we detected high amounts of labeled cells in the hippocampus, the entorhinal cortex and piriform cortex. In somewhat lower amounts, stained cells could be found in several nuclei of the amygdala (in the basomedial, basolateral, lateral, central and medial nucleus of the amygdala, in the amygdalopiriform transition area and in the amygdalohippocampal transition area as well as in the bed nucleus of the stria terminalis).     

Localization of angiotensin II AT1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata

There exist at least two distinct subtypes of angiotensin II receptors in the brain, namely the AT1 and AT2 subtypes. The high density of angiotensin II AT1 receptors is present in the medulla oblongata. The AT1 subtype of angiotensin II receptors mainly mediates central cardiovascular events. In the present study a polyclonal antibody against the angiotensin II AT1 receptor and a monoclonal antibody against tyrosine hydroxylase were employed to evaluate the possible presence of angiotensin II AT1 receptor-like immunoreactivity in the catecholaminergic neurons of the rat medulla oblongata by means of the double colour immunofluorescence technique. A weak, diffuse cytoplasmic angiotensin II AT1 receptor-like immunoreactivity was observed in almost all the catecholaminergic cell bodies of the A2, C1, C2 and C3 cell groups, except those of the A1 cell group containing moderately intense, diffuse cytoplasmic angiotensin II AT1 receptor-like immunoreactivity, occasionally found in the noradrenergic dendrites of the A1 cell group. There was a higher density of the angiotensin II AT1 receptor-like immunoreactive profiles in the A2 cell group area than in other catecholaminergic cell group areas. In addition, the angiotensin II AT1 receptor-like immunoreactivity was seen in non-catecholaminergic neurons. The present results provide evidence for the existence of the specific angiotensin II AT1 receptor-like immunoreactivity in the noradrenergic and adrenergic neurons of the rat medulla oblongata known to have a cardiovascular role. Thus, the findings support the view that angiotensin II AT1 receptors in the medulla oblongata participate in cardiovascular control and indicate a cellular substrate for the documented interaction between the angiotensin II and adrenergic transmission lines in cardiovascular function at the level of the nucleus tractus solitarii.     

Reproductive hormones modulate angiotensin II AT1 receptors in the dorsomedial arcuate nucleus of the female rat

Angiotensin II AT1 receptors are highly localized in the dorsomedial arcuate nucleus. AT1 receptor number is very low during proestrus and in ovariectomized and male rats, and is high only during the estrus phase of the estrous cycle and after ovariectomized rats receive a sequential estrogen-progesterone treatment. Our results suggest that the mechanism of the estrogen-progesterone inhibition of the prolactin surge may involve the selective stimulation of dorsomedial arcuate AT1 receptors.     

Blockade of angiotensin converting enzyme but not of angiotensin AT1 receptors improves glucose tolerance

The establishment of community-controlled organisations as part of the Aboriginal self-determination movement in Australia has failed to deliver the expected social and health gains for the majority of indigenous people. Compared with that of indigenous people in New Zealand, The United States and Canada, Aboriginal health has shown little improvement over the past two decades. There is strong evidence to suggest that universal education is itself a tool for liberation and also that educational attainment translates directly into better health. Nevertheless, attempts to account for the continuing poor health of indigenous Australians has continued to overlook the role of education in explaining health status differentials. This is not an attempt to undervalue the significant achievements of the Aboriginal self-determination movement. Rather, it is an attempt to draw attention to lack of educational attainment as a contradiction in the indigenous struggle for self-determination and better health. Based on existing data, as well as personal observations and discussions, this preliminary investigation seeks to draw attention to the lack of formal education as a barrier to Aboriginal social and health improvement. Public health practitioners and policy makers are called on to consider working collaboratively with Aboriginal communities to ensure that formal education is not only popularised but also that governments are made accountable for their constitutional responsibilities towards Aboriginal education.     

Localization of AT2 angiotensin II receptor gene expression in rat brain by in situ hybridization histochemistry

To localize the gene expression of AT2 angiotensin II receptors in rat brain we performed in situ hybridization histochemistry using 35S-labeled antisense riboprobes. The AT2 receptor mRNA expression pattern was compared in consecutive brain sections, from 2 week old rats, with the receptor expression by means of [125I]Sar1-ANG II binding and displacement with AT2 selective ligands followed by autoradiography. Expression of AT2 receptor mRNA was found in several thalamic nuclei (ventral posterolateral, mediodorsal, central medial, paracentral, and paraventricular), the medial geniculate nuclei, the nucleus of the optic tract, the subthalamic nucleus, the interposed nucleus of the cerebellum, and in the inferior olive. In these areas the AT2 receptor gene expression corresponds well with [125I]Sar1-ANG II binding. In addition, AT2 receptor mRNA expression was found in the red nucleus where no [125I]Sar1-ANG II binding was present. No significant hybridization of the AT2 receptor antisense probe was found in septal nuclei, the locus coeruleus, the dorsolateral geniculate nucleus, or the cerebellar cortex, areas rich in [125I]Sar1-ANG II binding. Our results indicate that some brain regions may be involved in AT2 receptor formation, transporting the receptor protein to other brain areas. However, in most structures, both the formation and expression of receptors occur, suggesting the existence of local AT2 receptor circuits, or that of AT2 autoreceptors. Other structures express only the receptor protein, indicating that these AT2 receptors are produced elsewhere. Our present data are the basis for further studies on the clarification of AT2 receptor pathways in the brain.     

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.     

Upregulation of angiotensin II AT1 receptors in the mouse nucleus accumbens by chronic haloperidol treatment

The distribution of angiotensin II AT1 and AT2 receptor subtypes were mapped in the mouse brain by in vitro autoradiography. Along with a differing distribution of AT1 and AT2 receptors in the hind brain compared to the rat, moderate densities of AT1 receptors were observed in dopamine-rich regions, namely the caudate putamen and nucleus accumbens, previously observed in the human, but not rat or rabbit. Considering our previous anatomical and functional studies demonstrating an interaction between brain angiotensin II and dopaminergic systems, the effect of chronic treatment with the dopamine antagonist, haloperidol, on AT1 and AT2 receptor levels was investigated in the mouse brain. Haloperidol treatment for 21 days resulted in an increase in angiotensin II AT1 receptor levels in the nucleus accumbens, accompanied by an increase in dopamine D2 receptors, but no change in dopamine D1 receptors. Striatal AT1 receptors did not alter with treatment, nor did AT1 or AT2 receptors in a number of brain regions not associated with dopaminergic systems, such as the median preoptic nucleus, paraventricular hypothalamic nucleus, and nucleus of the solitary tract. The present study suggests that brain angiotensin II-dopamine interactions extend beyond the known effects on the nigrostriatal dopaminergic system, to the mesocorticolimbic dopaminergic system.     

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P < 0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 mumol/100 g body weight as a bolus, i.v., plus a 30-min infusion of 0.018 mumol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P < 0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 +/- 0.28 mM, angiotensin II, N = 9 vs 6.4 +/- 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.     

Intracerebroventricular administration of angiotensin type 1 (AT1) receptor antisense oligonucleotides attenuate thirst in the rat

Chinese hamster ovary cells were pulse-labeled with 125I-iododeoxyuridine during early S phase, and cell samples were harvested 30 min or 5 h after labeling. The samples were frozen (with or without 25 mM cysteamine) and stored at -196 degrees C for accumulation of 125I decays. X-ray control experiments were performed at 37 degrees C and -196 degrees C. Aliquots of cells were plated for evaluating micronucleus formation and cell survival. The results demonstrated a striking shift in micronucleus formation and cell death with time after labeling. Cells frozen 30 min after labeling exhibited effects typical of low-LET radiation, but cells frozen 5 h after labeling showed a response characteristic of high-LET radiation. Cysteamine provided protection against the effects of 125I during the initial phase of effects characteristic of low-LET radiation, but no protection was seen during the phase characteristic of high-LET radiation. When cell survival was evaluated as a function of micronucleus frequency rather than dose in decays/cell, the survival curves for all treatment groups became superimposed. Previous work using the same experimental system had failed to show a direct link between 125I-induced DNA double-strand breaks and cell death. These findings are consistent with the hypothesis that DNA damage may not be the sole mechanism for cell killing and that damage to higher-order structures in the cell nucleus may contribute to (or modify) radiation-induced cell death.     

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.     

Function of the heart conduction system in Wolff-Parkinson-White syndrome

A study of the functional state of the heart conduction system by means of electrophysiologic testing of 46 patients with the Wolff-Parkinson-White syndrome has shown that the peculiarities of atrioventricular conduction and the development of an electrocardiographic picture in this disease are determined by the functions of anomalous accessory atrioventricular pathways--more frequently of the Kent's bundle and more infrequently of the Macheim's bundle. In addition, the degree of manifestations of the ventricular pre-excitation on the ECG depends on the correlation between impulse conduction times along the anomalous and normal pathways. The slow conduction along the Kent bundle form the basis of the latent WPW syndrome not recorded by the ECG.     

AT1 receptor-mediated stimulation by angiotensin II of rat aortic fibronectin gene expression in vivo

Fibronectin plays an important role in various vascular diseases. A subpressor (200 ng kg-1 min-1) or pressor (1000 ng kg-1 min-1) dose of angiotensin II was continuously infused into rats by osmotic minipump for various times, to investigate the effects on aortic fibronectin gene expression. In rats infused with a subpressor dose of angiotensin II in which blood pressure was normal for 3 days, aortic fibronectin mRNA levels started to increase by 1.4 fold at 12 h and reached the maximal levels (increased by 3.1 fold) at 3 days. Treatment with TCV-116 (3 mg kg-1 day-1), a non-peptide selective AT1 receptor antagonist, completely inhibited the angiotensin II-induced increase in aortic fibronectin mRNA, while hydralazine (10 mg kg-1 day-1) did not block this effect. Similar results were also obtained for a pressor dose of angiotensin II. Thus, angiotensin II directly stimulates aortic fibronectin gene expression in vivo, which is mediated by the AT1 receptor but not by blood pressure.     

Renal effects of AT1 angiotensin receptor antagonists (AT1ra)

RENIN-ANGIOTENSIN ANTAGONISTS: The renal effects of angiotensin II receptor antagonists (AT1 blockers) can be compared with another class of drugs inhibiting the renin-angiotensin-aldosterone system, i.e. the angiotensin I converting enzyme inhibitors (ACE1). SIMILAR BUT SPECIFIC EFFECTS: The renal effects of these two classes of drugs are similar but each class has specific effects explained by several mechanisms. i) The system includes a large number of active peptides (angiotensin II, angiotensin III, angiotensin 1-7) which exert various effects according to their specific receptor(s): ii) several types of angiotensin II receptors have been identified (AT1, AT2, AT4 ...). Only AT1 blockers are available in clinical practice. iii) Receptor or enzyme blockade can produce varying effects; ACE inhibition is not specific since increased bradykinin activity is associated with the suppression of angiotensin peptide generation. EXPERIMENTAL AND CLINICAL TRIALS: Experimental and recent clinical studies have shown that AT1 blockers can induce, like ACE1, hypotension, renal vasodilation and natriuresis. The definite effects on discrete renal structures (vessels, glomeruli, tubules) differ however in magnitude which may suggest specific indications according to the pathophysiological background (renal disease, congestive heart failure, etc.).     

Comparative pharmacology of recombinant rat AT1A, AT1B and human AT1 receptors expressed by transfected COS-M6 cells

1. Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes. 2. We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non-peptide AT1 receptor antagonist. Using a radioligand-receptor binding assay and membranes prepared from COS-M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes. 3. The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]-Sar1Ile8 angiotensin II for binding to the AT1 receptors. 4. Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences. 5. It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.     

Immunohistochemical study on the development of the rat heart conduction system using anti-Leu-7 antibody

Morphological aspects of the heart conduction system have been studied by various histochemical markers. However, the actual presence of markers in the conduction system and its developmental mode remain controversial. We have shown the anti-Leu-7 antibody to cross-react with cells of the rat embryonic heart conduction system by an immuno-electron microscopic study. A comparison was thus made between the results of Leu-7 immunohistochemistry and those by other markers previously used for studying the development of the conduction system. As a result, Leu-7 immunoreactivity proved the most reliable marker for studying the conduction system of the developing rat heart. Examination of the developmental mode of a rat conduction system was facilitated with the use of this marker. The immunoreactivity for Leu-7 initially appeared in the anterior wall of the bulboventricular region at 10 days of development. The development of three internodal tracts and two atrioventricular nodes was demonstrated in the following stage. These primordial atrioventricular nodes, one of which connects to a bundle of His, fused to form a single distinctive node followed by completion of the conduction system in the 18 day fetus.     

Increased immunoexpression of atrial natriuretic peptide in the heart conduction system of the rat after cardiac sympathectomy

The present investigation was designed to elucidate which role the sympathetic nerves play in the immunoexpression of atrial natriuretic peptide in the cardiac conduction system of the rat. In order to destroy the cardiac sympathetic nerve terminals, both surgical and chemical sympathectomy were performed. By use of immunohistochemical and radioimmunoassay techniques, the immunoreactivity and level of atrial natriuretic peptide in the conduction system and in the cardiac myocardium were determined. In contrast to the low degree of immunoreaction for atrial natriuretic peptide seen in control rats, the sympathectomized rats exhibited pronounced immunoreactivity for atrial natriuretic peptide in the atrioventricular bundle and bundle branches, which normally have high numbers of sympathetic nerve fibres. On the other hand, in the peripheral parts of the conduction system, where there are ordinarily few sympathetic nerve fibres, the degree of immunoreaction was unchanged. The quantitative measurements also showed that the entire ventricles, including the conduction system, contained increased levels of atrial natriuretic peptide in the treated hearts. The present study shows that destruction of the sympathetic nervous system leads to an increased level of atrial natriuretic peptide in the Purkinje fibres of bundle branches, which thus seem to have a dormant capacity for synthesis of this peptide. The results provide new evidence about the change in atrial natriuretic peptide levels that occurs when sympathetic innervation is altered.     

Contribution of nitric oxide to the acute antihypertensive effect of blockers of AT1 angiotensin receptors in spontaneously hypertensive rats

The acute vasodepressor effect of AT1 angiotensin receptor blockers losartan and CL329167 was compared in spontaneously hypertensive rats (SHR) pretreated and not pretreated with NG-monomethyl-L-arginine (LNMMA; 15 mg/kg i.v. bolus plus infusion at 10 mg/kg/h), an inhibitor of nitric oxide (NO) synthesis. The antihypertensive effect of losartan (30 mg/kg, i.v.) in SHR pretreated with LNMMA (-13 +/- 4 mmHg) was greatly diminished (P < 0.01) relative to the antihypertensive effect of losartan in SHR not pretreated with LNMMA (-44 +/- 8 mmHg). Similarly, the antihypertensive effect of CL329167 (5 mg/kg, i.v.) in SHR pretreated with LNMMA (-12 +/- 3 mmHg) was surpassed (P < 0.01) by the antihypertensive effect in SHR not pretreated with LNMMA. (-41 +/- 4 mmHg). However, pretreatment of SHR with LNMMA did not minimize the vasodepressor effect of prazosin, isoproterenol or sodium nitroprusside. The impairment in vasodepressor responsiveness to losartan in rats pretreated with LNMMA was not demonstrable in rats concurrently receiving sodium nitroprusside to correct for the loss of endogenous NO, or atrial natriuretic peptide which also increases vascular cGMP. These data suggest that a mechanism mediated by NO and/or cGMP is necessary for the full expression of the acute antihypertensive effect of AT1 angiotensin receptor blockers in SHR.     

Morphological studies on the human heart conduction system

An attempt has been made to demonstrate morphological equivalents of electrocardiographically determined conduction disturbances in the human heart. In 50% of our cases an authentic morphological change was found. In 25% the correlation remained doubtful, and in another 25% there was no connection. The highest coincidence was found in chronic left bundle branch block and in dissecting aneurysms causing hemopericardium. The correlation between function and morphology is very loose in patients who have undergone cardiac surgery. Etiology is discussed only marginally; ischemia is the predominant feature.     

Regulation of the angiotensin AT1 receptor expression by hypercholesterolemia

The AT1 receptor mediates most of the biological effects of angiotensin II and has therefore been implicated in the pathogenesis of hypertension as well as arteriosclerosis. Hypercholesterolemia is a prominent risk factor for the development of these cardiovascular diseases. Since experimental results from hyperlipidemic animal models suggested an interaction of the renin-angiotensin system and hypercholesterolemia, the effects of low-density lipoprotein (LDL) and hypercholesterolemia has recently been investigated in vitro and in vivo. LDL causes in cultured vascular smooth muscle cells an up-regulation of AT1 receptor gene expression which is followed by an enhanced functional response upon stimulation with angiotensin II. This effect is also evident in vivo, as assessed in hypercholesterolemic rabbits. The vascular AT1 receptor expression is increased approximately twofold in hypercholesterolemic rabbits in comparison to normocholesterolemic animals. This leads ultimately to an enhanced angiotensin II-induced vasoconstriction. Thus, hypercholesterolemia and elevated concentrations of LDL enhance AT1 receptor expression in the vasculature causing an enhanced biological effectiveness of the renin-angiotensin system. This interaction may explain that blockade of the renin-angiotensin system attenuates the progress of arteriosclerosis in the hypercholesterolemic organismen and indicates a pathophysiologically important role for the lipid-induced AT1 receptor up-regulation.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.