Specific Pregnancy-Induced Angiotensin II Type-1 Receptor Expression in Ovine Uterine Artery Does Not Involve Formation of Alternate Splice Variants or Alternate Promoter Usage¹

^c Departments of Obstetrics & Gynecology, Perinatal Research Laboratories, and ^d Meat & Animal Science, University of Wisconsin-Madison, Madison, Wisconsin 53715

	ABSTRACT

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Recently we reported that pregnancy is associated with a dramatic increase in angiotensin II type-1 receptor (AT₁-R; both protein and mRNA) in ovine uterine artery endothelial cells (UAEC), which far exceeds that seen in omental (systemic) arteries. Recent reports also suggest that alternate splicing of AT₁-R mRNA may play a role in regulation of AT₁-R expression in humans. Herein, we have investigated the possibility of alternate transcript splicing/promoter usage in UAEC from pregnant ewes by 5'-RACE (rapid amplification of cDNA 5'-ends). To provide our control "reference" sequences, we first performed 5'-RACE analysis of AT₁-R mRNA transcripts in liver, kidney, and adrenal cortex. Analysis of 17 resultant clones showed exceptional homology, indicating that a single identically spliced mRNA product is observed in all three ovine tissues. Homology of the 5'-untranslated region to that of the human was low (34.2%), but four in-context start/stop codons and the beginning of human exons 1 and 5 were highly conserved. Subsequently we isolated 30 individual clones using UAEC RNA from three pregnant ewes and found no evidence of any sequence formed through unique splicing or promoter usage. We conclude that the pregnancy-induced increase in AT₁-R expression unique to UAEC during pregnancy is not mediated by splicing of a unique transcript or unique promoter usage.

	INTRODUCTION

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During pregnancy, marked hemodynamic changes occur, resulting in a mild decrease in blood pressure and a dramatic increase in cardiac output [1]. This may largely result from decreased systemic vascular resistance and increased refractoriness to the vasoconstrictor effects of circulating angiotensin II (AII). The uterine artery demonstrates the highest degree of refractoriness to AII-stimulated vasoconstriction compared to the systemic vasculature [2], which is appropriate in view of the ever-increasing nutritional and oxygen needs of the developing fetus. Angiotensin II type-I receptor (AT₁-R) levels on the vascular smooth muscle cells of uterine artery are unusually low compared to those of the systemic vasculature and remain low throughout pregnancy [3–5], but we have also recently shown a dramatic (8- to 10-fold) increase in expression of AT₁-R on uterine artery endothelial cells (UAEC) by the third trimester of pregnancy [6]. Activation of these endothelial AT₁-R by elevated levels of AII thus stimulates the increased generation of the endothelium-derived vasodilators nitric oxide (NO) and prostacyclin [3, 7, 8], which subsequently diffuse to the smooth muscle and inhibit vasoconstriction through the cGMP and cAMP signaling pathways, respectively [8].

Cloning of the AT₁ receptor from several species has now made it possible to study the molecular mechanisms underlying hormonal regulation of AT₁-R expression [9, 10]. The results of such studies in human as well as rodent tissues have suggested that expression of this receptor is hormonally controlled by many factors [9], but resultant changes in AT₁-R protein expression are generally related directly to changes in cellular AT₁-R mRNA levels [6, 11–14]. An understanding of the mechanisms underlying control of AT₁-R mRNA transcription are complicated still further in rats and mice by the description of two genes, denoted as AT_1A and AT_1B, which show tissue-dependent expression, i.e., rat adrenal expresses predominantly AT_1b whereas kidney expresses predominantly AT_1a mRNA, and liver expresses both [15]. Recently, putative human AT_1a- and AT_1b-like mRNA sequences were also reported [16], but, unlike the findings in rodents, the bulk of genomic Southern analysis published to date predicts only a single gene in the human and other mammalian species [9, 17], including sheep [18], and a second human AT₁ gene has neither been identified nor sequenced. Nevertheless, more detailed sequence analysis of AT₁-R mRNA variants in different human tissues, particularly liver and lung [19–21], suggests that alternate sequences are indeed present, and derive from complex alternate splicing of exons 1–4 of a single gene product to yield different 5'-untranslated regions, which are linked in turn to a common exon 5 encoding both the protein coding sequence and 3'-untranslated sequence [19–21]. Furthermore, the existence of such variants may have significant physiological relevance, since recent studies suggest that human AT₁-R mRNA variants are translated in vitro with different efficiencies according to whether their 5'-untranslated sequences contain false start/stop codon combinations and/or GC-rich regions [20, 21]. Although there are no instances reported of tissue-specific expression of any single variant, the relative levels of each variant do show marked differences between tissues [19, 21].

While tissue-specific expression of alternately spliced AT₁-R mRNA transcripts has been demonstrated in humans, the importance of alternate splicing events in control of AT₁-R expression in other mammalian species expressing one gene, such as the sheep, remains unknown. Nevertheless, the sheep is extensively used as the physiologic model for the human to study the role of AT₁-R in the control of vascular resistance, due to their comparable blood volume and vascular responsiveness to infused AII [2, 22]. Furthermore, although we have shown by reverse transcription-polymerase chain reaction (RT-PCR) that the pregnancy-induced expression of AT₁-R in uterine artery endothelium is associated with an increase in at least one transcript of AT₁-R mRNA [6], it is not clear if alternate transcripts are also generated at this time, either as a consequence of alternate splicing or promoter usage. Therefore, in this study, we first used the known partial protein coding sequence for ovine AT₁-cDNA [18] to clone the 5'-untranslated region of AT₁-R mRNAs from liver, kidney, and adrenal cortex poly(A)⁺ RNA to obtain reference ovine sequence data comparable to that published for the human [19–21]. (Adrenal was chosen because the previously published bovine AT₁-R cDNA sequence was also isolated from adrenal cortex [23]. Liver was chosen since multiple alternately spliced mRNA variants were reported in human liver [19, 20]. Kidney was used as a "control" to allow for the possibility of two AT₁-R genes since the putative human AT_1b sequence was reported to be expressed in liver, but not in kidney [16].) Then we tested the hypothesis that the pregnancy-induced increase in AT₁-R expression in uterine artery endothelial cells is largely due to additional tissue-specific formation of unique AT₁-R mRNA variants (i.e., different from those seen in liver, kidney, and adrenal cortex) through alternate splicing of the 5'-untranslated region and/or promoter usage.

	MATERIALS AND METHODS

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Isolation of RNA

Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences, and follow the recommended American Veterinary Medicine Association guidelines for humane treatment and euthanasia of laboratory farm animals. Ewes were subjected to nonsurvival surgery using i.v. general anesthesia (sodium [Na⁺] pentobarbital; Nembutal; 25–50 mg/kg), which was titrated in order to maintain tissue perfusion and oxygenation during the time of tissue collection. During nonsurvival surgery, liver, kidney, and adrenal were rapidly (5 min) obtained for study in ice-cold PBS (10 mM PBS is 8 mM Na₂HPO₄, 2 mM KH₂PO₄, 150 mM NaCl, pH = 7.4). Cubes (2–3 mm) of liver, kidney cortex, and adrenal cortex were dissected and snap-frozen in liquid nitrogen before RNA extraction. Total RNA was extracted from one cube of tissue by homogenization in guanidinium isothiocyanate/phenol/chloroform extraction. Briefly, tissue was homogenized in 4 ml RNAzol B (Cinna Biotecx, Houston, TX) and then dispensed into 4 x 1 ml-volumes. Phase separation was achieved by addition of 150 µl chloroform to each tube and centrifugation (12 000 x g, 30 min). The upper aqueous phase was extracted twice with phenol chloroform isoamyl alcohol in the presence of heavy-grade phase-lock gel (5 Prime3 Prime, Boulder, CO) before precipitation in an equal volume of isopropanol (-20°C, 1 h) and recovery by centrifugation. Poly(A)⁺ RNA was then purified from 3 tubes of the recovered RNA by passing over oligo(dT) columns (5 Prime3 Prime), thus retaining poly(A)⁺ RNA, exactly as described by the manufacturer. Recovery of poly(A)⁺ RNA, calculated relative to the remaining tube of total RNA, was routinely found to be approximately 5%.

UAEC were first isolated by collagenase digestion from 4 pregnant ewes (110, 120, 130, 142 days of gestation, spanning the third trimester), as described [6]. We have previously shown that cells isolated in this way are endothelial in origin and of high viability and purity, as demonstrated by their uniform expression of endothelial NO synthase, an endothelial marker in the uterine artery [24], their cobblestone morphology in primary culture, and their uptake of acetylated low-density lipoprotein as visualized under UV excitation [25]. For extraction of total cellular RNA, freshly isolated endothelial cells were washed in fresh M199 and pelleted by centrifugation before solubilizing in 1 ml RNAzol B (Cinna Biotecx). After addition of 150 µl chloroform and phase separation by centrifugation (12 000 x g, 20 min), the upper aqueous phase was removed, extracted twice with phenol/chloroform/isoamyl alcohol using heavy-grade phase-lock gel (5 Prime3 Prime), and finally mixed with 110% by volume of isopropanol. RNA was then precipitated by standing the mixture at -20°C for 1 h before recovery by centrifugation (12 000 x g, 30 min) and washing of the pellet in 75% ethanol. RNA was then solubilized in molecular biology-grade water (5 Prime3 Prime) and quantified by spectrophotometry before storage at -70°C.

5'-RACE Amplification

5'-RACE cDNA was prepared as suggested by the manufacturer using a 5'-RACE kit (Gibco, Gaithersburg, MD), together with oligonucleotides GSP1 (5'-GCG GTG GAT TAT AGT TGG-3') and GSP2 (5'-GGT GAA CAA TAG CCA GGT-3'), which are complementary to the protein coding region partially cloned previously by Robillard et al. [18], and corresponding to positions +433 and +329, respectively (positions relative to ATG start site; see below). Briefly, reverse transcription was performed using GSP1 and 0.5 µg poly(A)⁺ RNA each from liver, kidney cortex, and adrenal cortex at 42°C for 30 min; this was followed by RNAse digestion of the product and spin column recovery of the cDNA. 3'-Tailing of the cDNA with poly-dCTP was achieved as described, and subsequent C-tailed cDNA was specifically amplified using GSP2 and the abridged anchor primer (5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3') through 35 cycles with a 60°C annealing temperature and 1.5-min extension time. Subsequent reamplification of 5 µl of the first-round amplification product was performed with GSP2 and the abridged universal amplification primer (5'-GGC CAC GCG TCG ACT AGT AC-3') for 30 cycles under the same conditions. Note that 5'-RACE amplification as described was validated by including controls for each tissue, which were carried out in the absence of reverse transcriptase (RT-) or absence of dCTP tailing enzyme (dC-) in order to authenticate the results as mRNA-specific.

5'-RACE amplification of uterine artery endothelial RNA was performed by a procedure similar to that described, except that total RNA (0.5 µg/tube) was used as starting material.

Detection of RACE Products

RACE amplification products (10 µl each) were resolved in a 1% agarose/Tris glacial acetic acid EDTA (TAE) gel, vacuum-transferred (Bio-Rad Model 785 Vacuum Blotter) at 7 psi for 90 min to Magna NT (MSI, Westboro, MA) nylon membrane, and cross-linked by exposure to 150 mJ UV radiation (GS Gene Linker; Bio-Rad, Melville, NY). Hybridization membrane was prehybridized in a 20-ml volume of 5-strength saline sodium citrate (SSC; 20-strength SSC = 3.0 M NaCl + 0.3 M trisodium citrate, pH 7.0), single-strength modified Denhardt's solution (5-strength = 250 mM Tris-HCl pH 7.5, 0.5% sodium pyrophosphate, 5% SDS, 1% polyvinylpyrrolidone, 1% ficoll, 25 mM EDTA, and 1% BSA), 50% formamide, and 50 µg/ml tRNA using a Techne (Princeton, NJ) HB-1D hybridization oven at 42°C for 8 h. AT₁-R probe (20 x 10⁶ disintegrations per minute) generated by asymmetric PCR against a partial cDNA encoding the protein coding region of bovine AT₁-R [11, 26] was added, and hybridization continued overnight. The membrane was then washed once for 15 min at 30°C with double-strength SSC/0.1% SDS and twice for 30 min each at 30°C with 0.1-strength SSC/0.1% SDS, before exposure to MP Hyperfilm (Amersham, Arlington Heights, IL) for 15 min.

Cloning of Products

RACE amplification products (1 µl) were directly ligated into pCRII (TA Cloning kit; Invitrogen, San Diego, CA), and competent Escherichia coli was transformed with ligated plasmid using standard procedures. Colonies of transformed cells were grown on ampicillin/LB plates, and 24 clones for each tissue were selected by blue/white color selection and grown in miniculture. Recovered plasmids containing AT₁-R sequence were subsequently identified by restriction digest (EcoRI) and separated on a 1% agarose/TAE gel before transfer to hybridization membrane and hybridization against a probe encoding only the protein coding region (which overlaps the 3' end of all correctly generated RACE clones), as described above.

Sequencing

The plasmid inserts were sequenced from the 5' and 3' end using the PCR cycle sequencing/dideoxynucleotide termination method (Perkin Elmer, Foster City, CA) in the presence of [³³P]dCTP. Both 5'-RACE clones A7 and A19 were chosen for double-stranded sequencing throughout because of high recoveries and maximal length in each case. Otherwise, clones were single-stranded-sequenced except when resulting sequence diverged from that in A7 and A19. Consensus sequences were generated for 5'-RACE clones by contiguous alignment (WDNASIS; Hitachi, San Francisco, CA).

Primer Extension Analysis of AT₁-R mRNA 5'-Terminus

Ten picomoles OAT1R13 (5'-GGT AGG TAG GCA GAG CTG-3'), complementary to bases 63–80 of the consensus 5' RACE sense sequence, were end-labeled in the presence of 30 µCi [-³²P]dATP (3000 Ci/mmol) using T4 polynucleotide kinase as prescribed in the "Primer Extension System" from Promega (Madison, WI). Poly(A)⁺ RNA (13.4 µg) was pelleted into a single 0.5-ml thin-walled tube and solubilized in 5 µl molecular biology-grade water by heating to 65°C under mineral oil for 5 min. One hundred femtomoles end-labeled OAT1R13 and avian myeloblastosis virus (AMV) reverse transcriptase buffer were mixed with the poly(A)⁺ RNA (as well as a parallel water blank in a separate tube [-ve Con]), and contents were brought to 71°C before ramping down at 1°C/min to 42°C (to minimize mispriming). Reaction contents were equilibrated to room temperature for 10 min; then a master mix containing AMV reverse transcriptase and dNTPs was added to each tube. Reverse transcription was performed at 42°C for 30 min, after which 20 µl loading dye was finally added to each tube. Samples were then heated to 90°C for 10 min immediately before electrophoresis using a 38 x 50-cm SequiGen II sequencing rig (Bio-Rad, Hercules, CA). Twenty-microliter volumes of adrenal poly(A)⁺ RNA primer extension product, the parallel water blank (-ve Con), the kit +ve control primer extension product (run in parallel), and diluted X174HinfI end-labeled markers were loaded in adjacent lanes of a 6% polyacrylamide gel (preheated to 55°C) and resolved for 65 min at 2000 V. The vacuum-dried gel was exposed to Amersham MP Hyperfilm for 19 days.

	RESULTS

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5'-RACE amplification of AT₁-R mRNA from liver, kidney, and adrenal cortex successfully produced AT₁-R cDNA in both first- and second-round amplifications (not shown). A single predominant band was observed at ~720 base pairs (bp) for each tissue in both cases. No product of the same size was detected in reactions that either lacked reverse transcriptase (RT-) or lacked tailing enzyme (dC-), confirming that the products were derived from mRNA. The product size was consistent with that predicted, i.e., greater than 329 bases (since the 3'-terminal primer for 5'-RACE, GSP2, encoded at position 329 of the protein coding sequence by homology to the bovine sequence and less than ~1000 bases (by homology to the 5'-untranslated region + 329 bases identified in other known species).

Ovine 5'-RACE clone sequences isolated from liver, kidney cortex, and adrenal cortex showed > 99% homology of sequence, with no evidence of alternate splicing. The consensus sequence, also identical to clone A19, is shown in Figure 1. The size and homology of all 5'-RACE clones relative to the adrenal clone A19 are summarized in Table 1. Some showed a truncated 5' sequence, indicative of incomplete amplification since there was no consensus for the 5'-terminus between shorter clones, and clone L17 (liver) began 80 bases into the protein coding sequence. Whereas homology of the protein coding sequence was exceptionally high relative to the human cDNA, homology of the 5'-untranslated region was low (34.2%). Nevertheless, the intervening ovine sequence still contained four additional ATG sequences that were all close to corresponding "in frame" TGA stop codons. This repeated start/stop process has previously been shown in certain human AT₁-R mRNA variants to impair translation of AT₁-R in vitro [21]. In addition, the beginning of the exon 1 sequence and the beginning of exon 5 in the human are also highly conserved and in context in the ovine cDNA (Fig. 1).

View larger version (68K): [in this window] [in a new window]   FIG. 1. Consensus sequence of AT1-R cDNA 5'-RACE clones (GenBank accession number AF056308). The protein coding sequence is indicated by upper-case and the untranslated region by lower-case type. Repeated pairs of start/stop codons are indicated as bold/italic text (start) and bold/italic/underlined text (stop). Also shown is an alignment of the homologous regions at the beginning of human exon 1 (HEX1SU) and human exon 5 (HEX5GUO). Homologous bases are highlighted in bold type. The overlapping portion of the protein coding region detected by the bovine cDNA probe is underlined.

Primer extension studies were performed on adrenal cortex poly(A)⁺ RNA, the richest source of AT₁-R mRNA, in order to identify the 5'-terminus and allow comparison to the 5'-terminus of the 5' RACE clones. A single positive signal was detected specific to the poly(A)⁺ RNA lane and at 80 bases from the primer, the same distance corresponding to the 5'-terminus of the A19 RACE clones (Fig. 2).

View larger version (72K): [in this window] [in a new window]   FIG. 2. Primer extension analysis of ovine adrenal AT1-R mRNA 5'-terminus. Primer extension was performed as described using 13.4 µg poly(A)+ adrenal RNA, as described. A single predominant signal was detected in the adrenal poly(A)+ lane at the expected size of 80 bases (solid arrow), while the water control (-ve Con) gave no signal. The positive control signal (87 bases) derived using the kit control primer and RNA (+ve Con) is shown by the open arrow.

5'-RACE clones were isolated from UAEC using similar techniques for UAEC RNA from 3 of 4 pregnant ewes. Hybridizable signal at 720 bases was clearly observed by Southern blotting (not shown). No product was seen in the absence of reverse transcriptase for two of the three animals, with some faint signal present for the third. Sequence analysis of 10 clones each from the three animals (30 clones total) revealed once again a highly consistent sequence of > 99.7% homology to the previously isolated A19 clone (Table 2) and no evidence of alternate splicing or alternate promoter usage in the 5'-untranslated region. In addition, every UAEC 5'-RACE clone showed a 5'-terminus consistent with the adrenal RNA primer extension data. This lack of alternate splicing or alternate promoter usage was particularly surprising since pregnancy is a time of dramatically increased AT₁-R expression in UAEC [6], and yet the 5'-untranslated region of the UAEC 5'-RACE clones maintains the four premature start/stop combinations that could reduce translational efficiency [21].

While an attempt was also made to acquire similar products from UAEC RNA recovered from nonpregnant ewes (n = 4), no products were formed in these cases. However, we have previously shown by RT-PCR that the transcript isolated by 5'-RACE herein from pregnant ewes was also expressed in UAEC from nonpregnant ewes, albeit at a lower level [6].

	DISCUSSION

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Unlike results previously described in the human, the most striking features of the ovine 5'-RACE clone sequences isolated from liver, kidney cortex, and adrenal cortex were the very little divergence of sequence and the lack of evidence for the alternate splicing previously reported in the human [19–21]. Primer extension confirmed that the 5'-RACE clones were complete to the RNA terminus, and this finding was further supported by an extensive comparison to human cDNA and gene sequences, which showed the first 10 bases of the ovine AT₁-R cDNA to be 100% homologous to the beginning of exon 1 in the human [19]. Without the knowledge of genomic sequence, it is difficult to ascertain whether the ovine AT₁ gene is intronless, which would give one explanation for the lack of alternate splicing. While the overall homology of the 5'-untranslated region between human and sheep is low (34.2%), however, the beginning of exon 5 in the human is also highly conserved and in context in the ovine cDNA (Fig. 1), together with a putative splice site (CAGGT) at position 273. Thus, evidence for the preservation of at least one 5'-untranslated region exon boundary exists. We are currently in the process of isolating the ovine AT₁ gene in order to definitively locate these boundaries.

Our 5'-RACE cloning studies in UAEC from three pregnant ewes also concur with those from "nonreproductive" tissues (liver, kidney, and adrenal), and show for the first time that there is no evidence of novel AT₁-R mRNA splice variants or an alternate exon 1 sequence. This implies that the dramatic up-regulation of AT₁-R expression seen in uterine artery endothelium during pregnancy is not driven from a unique promoter or due to formation of transcripts of higher translational efficiency. Thus the pregnancy-induced increase in UAEC AT₁-R expression is controlled solely by altering the level of a single mRNA species, which in turn suggests that the predominant driving force during pregnancy must be a change in endocrine environment. Circulating estrogen [27] and AII [4, 28, 29] are both elevated in pregnancy, and the finding that AT₁-R gene regulatory elements include a growth factor (epidermal growth factor [EGF])-responsive consensus sequence [30] also raises the possibility for regulation by locally increased basic fibroblast growth factor, EGF, or vascular endothelial growth factor [31–33], as well as by sheer stress due to elevations in uterine blood flow [34, 35]. It is also possible that regulation by growth factors and estrogen could be functionally integrated at the level of the estrogen receptor itself since the estrogen receptor is a substrate for certain growth factor-activated kinases and can be activated independently of ligand by phosphorylation [36, 37]. A full understanding of these issues will ultimately require isolation of the ovine AT₁-R gene and its associated regulatory elements, for functional expression studies in cultured UAEC, which we are attempting at this time.

In conclusion, the previously reported alternate splicing of the 5'-untranslated region exons 1–4 in the human are not a general feature of AT₁-R mRNA processing in the sheep. Furthermore, the pregnancy-associated increase in AT₁-R expression in ovine uterine artery endothelium appears to be due to an increase in a single predominant transcript, rather than to the introduction of unique transcripts of higher translational efficiency by alternate splicing or promoter usage. The future understanding of this localized pregnancy-induced response, therefore, lies both in identification of the endocrine/paracrine regulators of this response and elucidation of the molecular mechanisms that mediate their actions. By these means, it may finally be possible to explore the molecular mechanisms that control normal pregnancy-induced flow increases through the uterine artery and also to propose a mechanistic basis for the failure of this adaptive response in conditions such as preeclampsia.

	ACKNOWLEDGMENTS

 
The authors would like to thank T.M. Phernetton for expert technical assistance and Dr. A.J. Conley (University of California, Davis) for constructive criticism of this study.

	FOOTNOTES

 
¹ These studies were supported by grants AHA-WI 95-GB-41, USDA 9601773, and NIH HL56702 to I.M.B. and HD33255, HL49210, and HL57653 to R.R.M.

² Correspondence: Ian M. Bird, University of Wisconsin-Madison, Department Obstetrics & Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 South Park St., Madison, WI 53715. FAX: (608) 257–1304; imbird{at}facstaff.wisc.edu

Accepted: March 9, 1998.

Received: November 24, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Magness RR, Zheng J. Maternal cardiovascular alterations during pregnancy. In: Gluckman PD, Heymann MA (eds.), Pediatric and Perinatal Perspectives: The Scientific Basis. London: Arnold Publishing; 1996: 762–772.
2. Naden RP, Rosenfeld CR. Effect of angiotensin II on uterine and systemic vasculature in pregnant sheep. J Clin Invest 1981; 68:468–474.
3. Cox BE, Rosenfeld CR, Kalinyak JE, Magness RR, Shaul PW. Tissue specific expression of vascular smooth muscle angiotensin II receptor subtypes during ovine pregnancy. Am J Physiol 1996; 271:H212–221.
4. Mackangee HR, Shaul PW, Magness RR, Rosenfeld CR. Angiotensin II vascular smooth-muscle receptors are not down-regulated in near-term pregnant sheep. Am J Obstet Gynecol 1991; 165:1641–1648.[Medline]
5. Bing C, Johnson IR, Pipkin FB. Angiotensin receptors in myometrium and myometrial vessels from uteri of women during the follicular and luteal phases of the menstrual cycle and in late pregnancy. Clin Sci 1996; 90:499–505.[Medline]
6. Bird IM, Zheng J, Cale JM, Magness RR. Pregnancy induces an increase in angiotensin II type-1 receptor expression in uterine but not systemic artery endothelium. Endocrinology 1997; 138:490–498.[Abstract/Free Full Text]
7. Magness RR, Rosenfeld CR. Calcium modulation of endothelium derived prostacyclin production in ovine pregnancy. Endocrinology 1993; 132:2445–2452.[Abstract/Free Full Text]
8. Magness RR, Rosenfeld CR, Hassan A, Shaul PW. Endothelial vasodilator production by uterine and systemic arteries. I. Effects of ANG II on PGI₂ and NO in pregnancy. Am J Physiol 1996; 270:H1914–1923.
9. Sandberg K. Structural analysis and regulation of angiotensin II receptors. Trends Endocrinol Metab 1994; 5:28–35.
10. Inagami T, Harris RC. Molecular insights into angiotensin II receptor subtypes. NIPS 1993; 8:215–235.[Abstract/Free Full Text]
11. Bird IM, Mason JI, Rainey WE. Regulation of type-1 angiotensin II receptor mRNA expression in human adrenocortical carcinoma H295 cells. Endocrinology 1994; 134:2468–2474.[Abstract/Free Full Text]
12. Lehoux JG, Bird IM, Rainey WE, Tremblay A, Ducharme L. Both low sodium and high potassium intake increase the level of adrenal angiotensin-II receptor type-1, but not that of adrenocorticotropin receptor. Endocrinology 1994; 134:776–782.[Abstract/Free Full Text]
13. Bird IM, Mason JI, Rainey WE. Regulation of angiotensin II type 1 receptor binding and AT1-R mRNA levels in human adrenocortical cells. Endocr Res 1995; 21:169–182.[Medline]
14. Bird IM, Word RA, Clyne C, Mason JI, Rainey WE. Potassium negatively regulates angiotensin II type 1 receptor expression in human adrenocortical H295R cells. Hypertension 1995; 25:1129–1134.[Abstract/Free Full Text]
15. Kakar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD. Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation. Biochem Biophys Res Commun 1992; 183:1090–1096.[CrossRef][Medline]
16. Konishi H, Kuroda S, Inada Y, Fujisawa Y. Novel subtype of human angiotensin II type-1 receptor: cDNA cloning and expression. Biochem Biophys Res Commun 1994; 199:467–474.[CrossRef][Medline]
17. Yoshida H, Kakuchi J, Guo DF, Furuta H, Iwai N, Meer-de Jong R, Inagami T, Ichikawa I. Analysis of the evolution of angiotensin II type 1 receptor gene in mammals (mouse, rat, bovine and human). Biochem Biophys Res Commun 1992; 186:1042–1049.[CrossRef][Medline]
18. Robillard JE, Schutte BC, Page WV, Fedderson JA, Porter CC, Segar JL. Ontogenic changes and regulation of renal angiotensin II type 1 receptor gene expression during fetal and newborn life. Pediatr Res 1994; 36:755–762.[Medline]
19. Su B, Martin MM, Beason KB, Miller PJ, Elton TS. The genomic organization and functional analysis of the promoter for the human angiotensin type 1 receptor. Biochem Biophys Res Commun 1994; 204:1039–1046.[CrossRef][Medline]
20. Guo DF, Furuta H, Mizukoshi M, Inagami T. The genomic organization of human angiotensin II type 1 receptor. Biochem Biophys Res Commun 1994; 200:313–319.[CrossRef][Medline]
21. Curnow KM, Pascoe L, Davies E, White PC, Corvol P, Clauser E. Alternatively spliced human type 1 angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms. Mol Endocrinol 1994; 9:1250–1262.[Abstract/Free Full Text]
22. Rosenfeld CR, Gant NF. The chronically instrumented ewe. A model for studying vascular reactivity to angiotensin II in pregnancy. J Clin Invest 1981; 67:486–492.
23. Sasaki K, Yamano Y, Bardham S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T. Cloning and expression of a complementary DNA encoding a bovine angiotensin II type-1 receptor. Nature 1991; 351:230–232.[CrossRef][Medline]
24. Magness RR, Shaw CE, Phernetton TM, Zheng J, Bird IM. Endothelial vasodilator production by uterine and systemic arteries II. Pregnancy effects on NO synthase expression. Am J Physiol 1997; 272:H1730-H1740.
25. Voyta JC, Netland PA, Via DP, Zetter BR. Specific labelling of endothelial cells using fluorescent acetylated low density lipoprotein. J Cell Biol 1984; 99:81A.
26. Millican DS, Bird IM. A general method for single stranded DNA probe generation. Anal Biochem 1997; 249:114–117.[CrossRef][Medline]
27. Carnegie JA, Robertson HA. Conjugated and unconjugated estrogens in fetal and maternal fluids of the pregnant ewe: a possible role for estrone sulfate during early pregnancy. Biol Reprod 1978; 19:202–211.[Abstract]
28. Magness RR, Cox K, Rosenfeld CR, Gant NF. Angiotensin II metabolic clearance rate and pressor responses in nonpregnant and pregnant women. Am J Obstet Gynecol 1994; 171:668–679.[Medline]
29. Naden RP, Coultrup S, Arant BS Jr, Rosenfeld CR. Metabolic clearance of angiotensin II in pregnant and nonpregnant sheep. Am J Physiol 1985; 249:E49-E55.
30. Guo DF, Inagami T. Epidermal growth factor-enhanced human angiotensin II type 1 receptor. Hypertension 1994; 23:1032–1035.[Abstract/Free Full Text]
31. Zheng J, Vagnoni KE, Bird IM, Magness RR. Expression of basic fibroblast growth factor, endothelial mitogenic activity and angiotensin II type-1 receptors in the ovine placenta during the third trimester of pregnancy. Biol Reprod 1997; 56:1189–1197.[Abstract]
32. Varner MW, Dildy GA, Hunter C, Dudley DJ, Clark SL, Mitchell MD. Amniotic fluid epidermal growth factor levels in normal and abnormal pregnancies. J Soc Gynecol Invest 1996; 3:12–19.[CrossRef][Medline]
33. Cheung CY, Singh M, Ebaugh MJ, Brace RA. Vascular endothelial growth factor gene expression in ovine placenta and fetal membranes. Am J Obstet Gynecol 1995; 173:753–759.[CrossRef][Medline]
34. Alshihabi SN, Chang YS, Frangos JA, Tarbell JM. Shear stress-induced release of PGE₂ and PGI₂ by vascular smooth muscle cells. Biochem Biophys Res Commun 1996; 224:808–814.[CrossRef][Medline]
35. Magness RR, Mitchell MD, Rosenfeld CR. Uteroplacental production of eicosanoids in ovine pregnancy. Prostaglandins 1990; 39:75–88.[CrossRef][Medline]
36. Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzy J, Couse JF, Korach KS. Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci USA 1996; 93:12626–12630.[Abstract/Free Full Text]
37. Bunone G, Briand PA, Miksicek RJ, Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 1996; 15:2174–2183.[Medline]