AT1B Receptor Research Articles

Recognition of tissue- and subtype-specific modulation of angiotensin II receptors using antibodies against AT1 and AT2 receptors.

Polyclonal antibodies have been prepared against synthetic peptides of human angiotensin II type-1 (AT1) and type-2 (AT2) receptors. Synthetic peptides corresponded to amino acids 15-24 of AT1 receptor and amino acids 241-253 of AT2 receptor. Western blot analysis of membranes from cell homogenates of COS-7 cells transfected with expression plasmids for mouse AT1b receptor, human vascular smooth muscle cells, and rat peripheral tissue demonstrated a major band of MW 44,000 with AT1 receptor antibody. Cell homogenates of COS-7 cells transfected with expression plasmids for human AT2 receptor showed a band of MW 44,000 with AT2 receptor antibody. Tissue homogenate of rat adrenal medulla and human pheochromocytoma presented a major band of MW 52,000 and a minor band of MW 44,000 with AT2 receptor antibody. AT1 receptor expression was in the following order: rat aorta > > lung, kidney, spleen, adrenal cortex > adrenal medulla, heart. Expression of AT2 receptor was in the following order: rat adrenal medulla > cortex, kidney, heart. Three-day treatment with CS866, an AT1 receptor antagonist, and temocapril, an angiotensin-converting enzyme inhibitor, suppressed AT1 receptor expression in the rat adrenal cortex, but not in the heart or adrenal medulla. AT2 expression was not affected by treatment with these drugs. These results suggest that newly developed antibodies for AT1 and AT2 receptors are useful in elucidating the regulation of subtype-specific receptor expression, and that AT1 but not AT2 receptors in adrenal tissue are regulated by angiotensin II.

Expression of angiotensin II receptor subtypes AT(1A) and AT(1B) in enriched fractions of dispersed rat pituitary cells.

Differential evaluation of angiotensin II (Ang II) receptors (AT1A, AT1B and AT2) expression was performed in dispersed adenohypophyseal cells fractionated by unit gravity sedimentation. Binding of [125I-Sar1-Ile8]-Ang II and its displacement by specific nonpeptidic AT1 (DuP753) and AT2 (PD123319) antagonists was monitored throughout the gradient. Quantification of mRNA levels corresponding to both AT1 receptor subtypes (AT1A and AT1B) was achieved by reverse transcriptase polymerase chain reaction (RT-PCR) amplification in the presence of an AT1 receptor mutant cRNA as internal standard. Fractions were characterised by radioimmunoassay for the five major anterior pituitary hormones and by counting immunocytochemically labelled cells. Quantification of AT1 receptor subtype mRNA levels was also performed in four hypophyseal cell lines secreting prolactin, growth hormone, corticotropin and a gonadotropin subunit. As already described for the whole pituitary, AT1B receptor mRNA is predominantly expressed (80% of total AT1A + AT1B receptor mRNA content), whereas AT1A is expressed at lower level (20%) in dispersed pituitary cells. Most AT1 receptor mRNA and binding co-elute with fractions enriched in lactotropes and corticotropes. In contrast to AT1B, AT1A receptor mRNA is not present in heavier populations of lactotropes or in somatomammotropes. Low AT1B mRNA levels are detected in GH4C1 and in GC cells, two clones which secrete respectively prolactin and growth hormone. In contrast, no AT1 receptor mRNA expression was found in two other cell lines, AtT20 and alphaT3-1, which produce pro-opiomelanocortin and gonadotropin. It is concluded that expression of AT1 receptor subtypes is heterogeneous in different populations of lactotropes and corticotropes.

Gene expression of central and peripheral renin-angiotensin system components upon dietary sodium intake in rats

The effects of dietary sodium intake on the gene expression of the renin-angiotensin system (RAS) were investigated in rat central and peripheral tissues in a single set of experiment. Northern and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to detect mRNA expression in rats fed a low- or a high-sodium diet (5 or 500 mmol Na +/kg diet) for 20 days. Plasma and renal renin levels were elevated in rats maintained on the low-sodium diet. Sodium deprivation enhanced the expression of angiotensinogen, renin, AT 1A and AT 1B receptor subtypes in the hypothalamus, but suppressed them in the brainstem. Kidney and adrenal levels of those mRNAs were also enhanced in the sodium-restricted rats. Both AT 1A and AT 1B mRNAs changed in a similar magnitude in each tissue examined upon dietary sodium intake. AT 1A was the predominant receptor subtype of AT 1 in all the tissues examined in the present study except the adrenal gland. The present study demonstrated that dietary sodium modulated the gene expression of the RAS components in the central and peripheral tissues. It also showed that the RAS components in the brainstem and hypothalamus were differentially expressed upon sodium deprivation. This suggests different roles of the RAS in these tissues in maintaining body fluid homeostasis in response to different sodium intakes.

Expression of AT1A and AT1B angiotensin II receptor messenger RNA in forebrain of 2-wk-old rats.

The gene expression of angiotensin II receptor subtypes AT1A and AT1B was localized in the forebrain of 2-wk-old rats by in situ hybridization histochemistry and compared with [125I]Sar1-angiotensin II binding patterns. AT1A receptor mRNA was expressed in circumventricular organs, in hypothalamic nuclei like the paraventricular nucleus, in the lateral olfactory tract, in the basolateral amygdaloid and anterior olfactory nuclei, and in the piriform cortex. No AT1B receptor mRNA was detected in these areas. AT1A and AT1B receptor mRNA was detected in the hippocampus, cingulate cortex, and choroid plexus. No forebrain area studied expressed AT1B receptor mRNA exclusively. Most often, a good match for receptor mRNA and binding was found. In some areas, apparent mismatches suggested receptor formation elsewhere (median eminence) or receptor presence in local neuronal circuits (hippocampus, cingulate, and piriform cortex). Our results support the hypothesis that AT1A receptors are involved in the classical central functions of angiotensin II. Both AT1A and AT1B receptors may play roles in cortical and limbic system function, particularly early in development.

Properties of AT1a and AT1b angiotensin receptors expressed in adrenocortical Y-1 cells.

Adrenocortical Y-1 cells were stably transfected with the AT1a and AT1b subtypes of the rat angiotensin (ANG)IIAT1 receptor cDNA to study the pharmacological and functional properties of the two receptors. Selected clones of transfected cells expressing the AT1a or AT1b receptor subtypes bound the native ligand ANG II and the peptide antagonist [Sar1,Ile8]ANG II with similar affinities, but they differed in their relative affinities for the nonpeptide antagonist losartan (half-maximal inhibitory concentration 9.7 and 4.7 nM), ANG III (126 and 33 nM), and the peptide antagonist [Sar1,Gly8]ANG II (6.2 and 1.2 nM). Photoaffinity labeling of the expressed receptors revealed a single component of 65 kDa for both receptor subtypes, suggesting that both receptors were glycosylated in a similar manner. The sensitivity of 125I-ANG II binding to AT1a and AT1b receptors to guanine nucleotides was unaffected by pertussis toxin treatment. ANG II stimulated the formation of inositol phosphates and increased the level of cytoplasmic Ca2+ in both At1a- and AT1b-transfected Y-1 cells. However, ANG II had little effect on forskolin-induced adenosine 3',5'-cyclic monophosphate accumulation, causing only minor inhibition in AT1a-transfected cells and slight enhancement in AT1b-transfected cells. These data indicate that AT1a and AT1b receptors show small but significant differences in their binding pharmacology and, upon activation, are coupled through Gq/G11 to the phosphoinositide-Ca2+ signaling pathway. However, neither AT1a nor AT1b receptors exhibit coupling to Gi and inhibition of adenylate cyclase when expressed in murine adrenal tumor cells.

Gene expression of angiotensin II receptor subtypes in the cerebellar cortex of young rats

Both AT1 and AT2 receptors were detected in the cerebellar cortex of 2-week-old rats by [125I]Sar1-angiotensin II binding. In contrast, using in situ hybridization histochemistry we found only AT1A and AT1B but not AT2 receptor mRNA in the cerebellar cortex of young rats. Expression of AT1A and AT1B receptor mRNA coding for either receptor subtype was found in the molecular or granular cell layers. Our results suggest that AT1 receptors may be located on dendrites of Purkinje cells expressing AT1A and AT1B receptor mRNA, whereas the AT2 receptor binding in the molecular layer may be located on nerve terminals of fibers projecting to the cerebellar, cortex from distant areas.

Angiotensin receptors and their therapeutic implications.

Angiotensin II is a multifunctional hormone that exerts its effects by interacting will cell surface receptors. Two major subtypes of receptors (AT1 and AT2) have been distinguished by pharmacological and molecular biological techniques. AT1 receptors have been further subdivided into AT1A and AT1B receptors. Several other isoforms have been found, notably in nonmammalian systems, but further information is necessary before definitive classification can be made. AT1 receptors mediate most known functions of angiotensin II, while AT2 receptors may be important developmentally. The molecular, structural, and biochemical characteristics of these receptors have been described, as well as the factors that regulate their expression. This receptor system has been implicated in several cardiovascular diseases, including hypertension, restenosis after angioplasty, cardiac hypertrophy, heart failure, myocardial infarction, and ventricular remodeling. Structural analysis of AT receptors may provide the basis for the development of new therapeutic agents with enhanced specificity for the treatment of these diseases.

Angiotensin II receptor gene expression in hypertrophied left ventricles of rat hearts

To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventricles of rats subjected to ventricular pressure overloading induced by aortic banding for 6 weeks and then 6 weeks medical treatment. Aortic banding was related to an increase in relative weight of the left ventricle from 1.73 +/- 0.06 (sham-operated) to 2.81 +/- 0.25 g/kg, an increase in beta-myosin: alpha-myosin messenger RNA (mRNA) ratio from 0.30 +/- 0.02 to 1.94 +/- 0.55 and an 18-fold increase in left ventricular atrial natriuretic peptide mRNA levels. In contrast, left ventricular pressure overload hypertrophy was not related to a significant change in the abundance of AT1a and AT1b mRNA, which were expressed in a relative ratio of 5:1. Similarly, the abundance of AT2 mRNA was not significantly changed in hypertrophied ventricles. In rats receiving the angiotensin II AT, receptor antagonist losartan (40 mg/kg) for 6 weeks after banding, relative heart weights were 2.39 +/- 0.14 g/kg, the beta-myosin: alpha-myosin ratio was 1.04 +/- 0.20 and atrial natriuretic peptide mRNA levels displayed a blunted increase (11-fold over sham-treated controls), documenting a significant amelioration of left ventricular hypertrophy by blockade of the AT1 receptor. Losartan treatment in parallel did not affect AT1a, AT1b and AT2 receptor mRNA levels, which were not different from those in vehicle-treated or sham-treated controls. These findings confirm that left ventricular hypertrophy in the rat is associated with increased ventricular expression of beta-myosin and of atrial natriuretic peptide and with reduced expression of alpha-myosin. Despite these significant changes in cardiac gene expression no alteration was observed in AT1a, AT1b and AT2 receptor mRNA levels.

Type 1 Angiotensin II Receptor Subtypes in Kidney of Normal and Salt-Sensitive Hypertensive Rats

We studied the localization and regulation of the two type 1 angiotensin II receptor subtypes AT(1A) and AT(1B) in different renal zones of the rat kidney by a reverse transcription-polymerase chain reaction amplification method. The yield of the reaction was quantified with an internal standard that was a 63-bp deleted mutant cRNA of the AT(1A) receptor. In kidneys of male Sprague-Dawley rats (n=4), the levels of AT(1A) and AT(1B) receptor mRNAs were highest in the inner stripe of the outer medulla, lowest in the inner medulla, and intermediate in the cortex and outer stripe of the outer medulla. Results (mean+/-SE) expressed in 10(5) molecules per microgram total RNA were for cortex outer stripe, inner stripe, and inner medulla, respectively, 171 +/- 15, 152 +/- 27, 322 +/- 10, and 73 +/- 3 for AT(1A), and 35 +/- 9, 26 +/- 1, 71 +/- 10, and 53 +/- 11 for AT(1B). In sabra rats sensitive (n=6) or resistant (n=6) to salt-induced hypertension and maintained on a normal salt diet, the percentage and level of each receptor subtype mRNA in cortex and outer stripe were similar in the two strains and comparable to those observed in Sprague-Dawley rats. However, AT(1A) of the inner stripe was significantly decreased in salt-resistant compared with salt-sensitive rats (166 +/- 28 and 318 +/- 58 10(5) molecules per microgram total RNA, respectively). These modifications were organ specific because no difference in the level of the receptor mRNAs was observed in the liver of the two Sabra rat strains, whereas a twofold increase in AT(1A) mRNA level but not in AT(1B) mRNA level was apparent in adrenal and in one renal zone, the inner stripe of the outer medulla, of hypertension-prone Sabra rats.

The developmental increase of the AT1A, but not the AT1B, receptor MRNA level at the preoptic area in spontaneously hypertensive rats

To determine the possible involvement of the central angiotensin system in hypertension, the angiotensin II type-1 receptor subtype mRNA levels in the preoptic area (POA) and hypothalamus were measured by means of a reverse-transcriptase/polymerase chain reaction in spontaneously hypertensive rats (SHR), and the results were then compared with the findings in age-matched normotensive Wistar-Kyoto rats (WKY). In 4-week-old (prehypertensive stage) and 7-week-old (evolving stage) SHR, the AT1A and AT1B receptor subtype mRNA levels in the POA and hypothalamus did not show any significant difference between the SHR and WKY. However, in the 16-week-old SHR (hypertensive stage), AT1A receptor subtype mRNA at POA was approximately 2-fold higher than in the WKY, while the AT1B receptor subtype mRNA showed no difference. On the other hand, neither the AT1A nor the AT1B mRNA level at the hypothalamus were different between the 16-week-old SHR and WKY. These results suggest that the AT1A mRNA level, but not the AT1B mRNA level, increases in the POA in hypertensive stage of SHR and the increase may therefore be related in some way to the state of hypertension.

Sodium induced regulation of angiotensin receptor 1A and 1B in rat kidney.

Two AT1 receptor subtypes, AT1A and AT1B, have been cloned and sequenced in the rat.15 Although AT1A and AT1B share a high degree (96%) of amino acid homology,3, 5 they may be functionally different. In studies of AT1A and AT1B receptors transfected into COS-7 cells,4 Sanderberg et al have shown that there are dose-related differences during angiotensin II (Ang II)-induced Ca2+ signalling and differences in binding affinity for Ang II agonists and antagonists between AT1A and AT1B. Although it has been noted that AT1 receptor subtypes in the rat brain can be differentially regulated by hormones or changes in dietary sodium,5, 6 gene regulation of the AT1 receptor subtypes in the kidney has not been fully investigated. Considering the key role of the kidney in the control of fluid homeostasis and blood pressure, it is important to understand the regulation of these two distinct receptor genes in the kidney. Such studies could lead to further insights into the relevance of these receptor genes in hypertensive disease states and possibly could lead to the development of new and more specific therapies. In this study, we used Northern blot and in situ hybridization analysis to test the hypothesis that dietary sodium intake differentially regulates gene expression of the AT1A and AT1B receptor subtypes in the kidney.

A functional comparison of the rat type-1 angiotensin II receptors (AT 1AR and AT 1BR)

To evaluate and functionally compare the rat AT 1A and AT 1B receptor subtypes, stable Chinese hamster ovary (CHO) cell lines expressing either recombinant receptor in approximately equal numbers were generated. Radioligand binding data suggests that the recombinant AT 1A receptor is pharmacologically similar to the recombinant AT 1B receptor. Functional studies indicate that both receptor subtypes can independently activate the phospholipase C IP 3 and the dihydropyridine-sensitive voltage-dependent Ca 2+ channel signal transduction pathways with equal efficiency, but are unable to modulate cAMP accumulation under our experimental conditions. Furthermore, both receptors can be directly involved in the cellular growth properties of AII. Slot-blot experiments clearly demonstrate that these receptors are expressed in a tissue-specific manner. A sequence comparison of the 5′ flanking regions of these two genes shows that they have very little sequence homology (∼ 36%), suggesting that although the AT 1A and AT 1B receptors appear to be pharmacologically and functionally similar, the control of their expression seems to be governed by distinct transcription factors.

AT1A, AT1B, and AT2 angiotensin II receptor subtype gene expression in rat brain.

We localized the gene expression of angiotensin II receptor subtypes (AT1A, AT1B, and AT2) in 2-week-old rat brain by in situ hybridization using subtype specific riboprobes. AT1A receptor mRNA but not AT1B or AT2 receptor mRNA was expressed in the subfornical organ and paraventricular nucleus of the hypothalamus. AT1B as well as AT1A receptor mRNA were found in the cerebral cortex and hippocampus. Conversely, AT2 receptor mRNA, but not AT1A or AT1B, was expressed in the medial geniculate nucleus and inferior olive. Our results indicate that AT1A receptors are involved in the well known central functions of angiotensin II. These results also support the hypothesis of the involvement of AT2 receptors in sensory and motor function.

Effects of des-Asp-angiotensin I on the electrically stimulated contraction of the rabbit pulmonary artery

In the presence of 3 × 10 −6 M captopril, 5 × 10 −7 M des-Asp-angiotensin I was found to inhibit the electrically (1 and 2 Hz) induced contraction of the rabbit pulmonary artery but had no significant effect on the noradrenaline-stimulated contraction. 2.8 × 10 −6 M indomethacin and 10 −6 M losartan but not 10 −6 M ( S) 1-{[4-(dimethylamino)-3-methylphenyl]methyl}-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1 H-imidazo{4,5- c]pyridine-6-carboxylic acid, ditrifluoroacetate, dihydrate (PD123319) attenuated the inhibition. The inhibition of the electrically stimulated contraction by 5 × 10 −7 M des-Asp-angiotensin I coincided with a significant drop in the accompanying evoked 3H overflow from re-uptaken [ 3H]noradrenaline. The results indicate that des-Asp-angiotensin I acts presynaptically on a subtype of angiotensin receptor that involves the release of prostaglandin(s). In addition, this receptor subtype is susceptible to blockade by angiotensin AT 1- but not AT 2-specific receptor antagonists. It was suggested that this receptor subtype is identifiable with the recently described angiotensin AT 1B receptor subtype found in the brain, pituitary and adrenal glomerulosa. These findings demonstrated a direct action of sub-micromolar concentrations of des-Asp-angiotensin I on a blood vessel and indicate that the nonapeptide is an active angiotensin per se.

Expression of cardiac angiotensin II AT1 receptor genes in rat hearts is regulated by steroids but not by angiotensin II

To examine the regulation by angiotensin II and by steroids of the expression of the angiotensin II AT1a and AT1b receptor genes in rat hearts. Endogenous levels of angiotensin II in the rats were increased either by unilateral 0.2-mm renal artery clips or by subcutaneous infusions of frusemide (12 mg/day) and by low-sodium diet. To inhibit endogenous angiotensin II actions the rats received the AT1 receptor antagonist losartan (40 mg/kg per day) or the angiotensin converting enzyme inhibitor ramipril (8 mg/kg per day). Circulating levels of glucocorticoids were elevated by subcutaneous injections of dexamethasone (400 micrograms/kg per day) and levels of mineralocorticoids were increased by subcutaneous injections of deoxycorticosterone acetate (2 mg/kg per day). AT1a and AT1b messenger RNA (mRNA) levels were semiquantified by reverse-transcriptase polymerase chain reaction and related to actin mRNA. The AT1a mRNA:AT1b mRNA ratio in the hearts of untreated rats was 10:1. Unilateral renal artery clipping led to a 30% decrease in AT1a mRNA, whereas treatment with frusemide, losartan or ramipril had no effect on the AT1a or AT1b mRNA levels. Rats fed a low-sodium diet showed a 37% increase in AT1a gene expression. Dexamethasone increased AT1a mRNA by 100% and AT1b mRNA by 300%, whereas deoxycorticosterone acetate treatment decreased AT1a mRNA levels to 30% of the control values. The present results suggest that the expression of the predominant cardiac AT1a receptor gene is not feedback-regulated by endogenous angiotensin II, whereas steroid hormones appear to be effective regulators, because glucocorticoids stimulate AT1 receptor gene expression and mineralocorticoids inhibit it.

The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control.

Subtypes of the angiotensin II (Ang II) type-1 (AT1) receptor are probably involved in distinct actions of the peptide, since their distribution in peripheral organs and regulation of their gene expression are different. We investigated the distribution of AT1A and AT1B receptor subtype mRNAs in the rat forebrain and pituitary using sensitive cRNA probes for in situ hybridization. High level of AT1A receptor mRNA expression is observed in the subfornical organ (SFO) and in the anterior hypothalamus, particularly the periventricular tissue surrounding the anterior portion of the 3rd ventricle (AV3V), which contains the organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus and the preoptic periventricular nucleus as well as in the hypothalamic periventricular nucleus and in the parvocellular part of the paraventricular nucleus (PVN). Moderate to strong AT1A labeling was found in the anterior olfactory nucleus, the piriform cortex and the nucleus of the lateral olfactory tract. Very low AT1B receptor mRNA expression was found in the SFO and the PVN. In contrast, strong AT1B receptor mRNA expression coincided with low AT1A receptor mRNA expression in the anterior pituitary. Labeling was cytoplasmic at the light microscopic level. We thus suggest that the AT1A receptor is responsible for the central actions of Ang II in the rat forebrain whereas direct actions of Ang II on the anterior pituitary are mediated by the AT1B receptor subtype.

Differential regulation of angiotensin II receptor subtypes in rat kidney by low dietary sodium.

This study was designed to determine whether expression of renal messenger RNA (mRNA) encoding the two known angiotensin II type 1 (AT1) receptor subtypes (AT1A and AT1B) can be regulated by dietary sodium. Seven-week-old male Wistar rats were fed a low-sodium diet (0.07%, n = 9) or a normal-sodium diet (0.5%, n = 9 [control]) for 14 days. A rat AT1 complementary DNA (cDNA) probe, which hybridizes to mRNA encoding both the AT1A and AT1B receptor subtypes, and cDNA probes, which are selective for AT1A or AT1B mRNA, were used in Northern blot or in situ hybridization analysis. By use of Northern blot analysis, renal mRNA levels for the AT1 and AT1A receptors in rats fed a low-sodium diet were found to be increased twofold (P < .05) compared with control. Because renal AT1B mRNA content was not detected by Northern blot analysis, quantitative image analysis of in situ hybridization with a digoxigenin-labeled cRNA probe made from AT1B cDNA was used. In situ hybridization analysis indicated that AT1B mRNA was expressed in the proximal and collecting tubules of the kidney in rats fed a normal-sodium diet. The low-sodium diet significantly decreased the percent positive staining area of AT1B mRNA in the renal cortex (5.51 +/- 0.77% versus 2.73 +/- 0.35%, P < .05) and medulla (4.76 +/- 0.70% versus 2.01 +/- 0.43%, P < .05) compared with the control diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of angiotensin II type 1 receptor subtype mRNA in rat kidney.

The physiological effects of angiotensin II (ANG II) on the kidney are mediated primarily by the ANG II type 1 (AT1) receptor. Two highly similar AT1 receptor subtypes have been identified in the rat by molecular cloning techniques, namely AT1A and AT1B. The intrarenal localization of the AT1A and AT1B receptor subtypes has not been studied by hybridization methods with subtype-specific receptor probes. Using radiolabeled probes from the 3' noncoding region of the AT1A and AT1B cDNAs, we localized AT1 mRNA in rat kidney by in situ hybridization. Specificity of the 3' noncoding region probes was tested by Northern blot and solution hybridization methods. AT1A mRNA levels were highest in the liver, kidney, and adrenal. In contrast, AT1B mRNA levels were highest in the adrenal and pituitary and low in kidney. Autoradiographic localization of 125I-[Sar1,Ile8]ANG II binding indicated that the highest levels of AT1 receptors were found in glomeruli and vascular elements. In situ hybridization with a nonselective AT1 receptor riboprobe indicated that the highest levels of AT1 mRNA were in the outer medullary vasa recta and cortical glomeruli with additional diffuse labeling of the cortex and outer medulla, consistent with labeling of tubular elements. In contrast, in situ hybridization with the AT1 subtype selective probes revealed that AT1A receptor mRNA was primarily localized to the vasa recta and diffusely to the outer stripe of the outer medulla and the renal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the angiotensin II receptor AT1B gene as a candidate gene for blood pressure

To answer two specific questions concerning the possible role played by the angiotensin II receptor AT1B (AT1B) locus in genetic hypertension. First, do alleles at the AT1B locus cosegregate with blood pressure in F2 populations in the rat and, if so, is this due to AT1B or a closely linked quantitative trait locus (QTL)? Secondly, are there any significant nucleotide differences between the coding regions of the AT1B gene of the inbred Dahl salt-sensitive (SS/Jr) strain and various control strains, or between the Lyon hypertensive (LH) strain the Lyon normotensive (LN) strain? The first objective was achieved by studying several F2 rat populations and by analyzing DNA markers around the AT1B locus. The second objective was accomplished by amplifying the AT1B coding regions by polymerase chain reaction from the genomic DNA, and by sequencing the relevant coding regions of the AT1B genes. In two F2 populations involving the SS/Jr rat, AT1B cosegregated only weakly with systolic blood pressure. Also, nucleotide differences resulting in conservative changes in three amino acids were detected between the coding region of the SS/Jr AT1B allele and the AT1B alleles of the several control strains. No nucleotide differences were found in the coding regions of the AT1B alleles between the LH and LN strains. There appeared to be a QTL which had a minor effect on blood pressure, located near the AT1B locus on rat chromosome 2. However, the AT1B gene itself is not supported as the candidate gene for causing blood pressure differences, because no nucleotide changes in the coding region that were functionally meaningful or concordant with the cosegregation analysis were detected.

The genomic organization of the rat angiotensin II receptor AT 1B

Expression of two subtypes of angiotensin type 1 receptor (AT 1A and AT 1B) seems to be controlled by different mechanisms. As a step toward the elucidation of difference in the mechanism in their gene expression, the genomic organization of rat angiotensin II type 1B receptor was investigated. Rat genomic DNA fragments were cloned by screening a genomic library with fragments from two cDNA clones (2.3 and 2.2 kb) encoding the rat AT 1B receptor. Three λ clones were isolated, and the hybridizing restriction fragments were sequenced. Comparison of the genomic clone DNA and cDNA sequences revealed that the rat AT 1B receptor gene contains three exons and two introns. Two of the exons encoded 5′ untranslated sequences while the third exon contained the entire coding region. The 5′-flanking region contained the typical sequence motifs found in many eukaryotic promoters including a TATA box and a potential SP1 binding site. The transcription initiation site was located at −8 bp upstream the 5′ end of the cDNA in the 5′-flanking region.