Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells.

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.

Inhibition of neointima formation by a nonpeptide alpha(v)beta(3) integrin receptor antagonist in a rabbit cuff model.

This study was performed to determine whether a highly selective nonpeptide alpha(v)beta(3) antagonist (SH306) would prove effective in inhibiting neointima formation in a rabbit cuff model. The animals were dosed with SH306, 5 mg/kg i.v., followed by 10 mg/kg s. c., 3 times daily for 3 days, or with vehicle (10% DMAC). Rabbits were sacrificed and perfused on days 1, 3, and 21; the vessels were paraffin embedded. A reduction in the intima/media (I/M) of the SH306-treated rabbits, as compared with the vehicle-treated control group, was noted (0.20 vs 0.36 [n = 4]). A significant increase in the area of the media was observed in the SH306-treated group versus the control group (0.20 vs 0.13). No difference was observed in cell proliferation between SH306 and vehicle after 1-day and 3-day dosing. Thrombi were found in 43% of the control vessels and in only 14% of the drug-treated vessels. No anticoagulant was used during the surgical procedure. No increase in inhibition of GPIIb/IIIa was observed in SH306-treated animals, as compared with the vehicle control group. We conclude that selective inhibition of alpha(v)beta(3) reduced neointima formation in a rabbit model at 3 weeks.

Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.

Dexfenfluramine was approved in the United States for long-term use as an appetite suppressant until it was reported to be associated with valvular heart disease. The valvular changes (myofibroblast proliferation) are histopathologically indistinguishable from those observed in carcinoid disease or after long-term exposure to 5-hydroxytryptamine (5-HT)(2)-preferring ergot drugs (ergotamine, methysergide). 5-HT(2) receptor stimulation is known to cause fibroblast mitogenesis, which could contribute to this lesion. To elucidate the mechanism of "fen-phen"-associated valvular lesions, we examined the interaction of fenfluramine and its metabolite norfenfluramine with 5-HT(2) receptor subtypes and examined the expression of these receptors in human and porcine heart valves. Fenfluramine binds weakly to 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. In contrast, norfenfluramine exhibited high affinity for 5-HT(2B) and 5-HT(2C) receptors and more moderate affinity for 5-HT(2A) receptors. In cells expressing recombinant 5-HT(2B) receptors, norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increased intracellular Ca(2+), and activated the mitogen-activated protein kinase cascade, the latter of which has been linked to mitogenic actions of the 5-HT(2B) receptor. The level of 5-HT(2B) and 5-HT(2A) receptor transcripts in heart valves was at least 300-fold higher than the levels of 5-HT(2C) receptor transcript, which were barely detectable. We propose that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or 5-HT released from carcinoid tumors (with or without accompanying 5-HT(2A) receptor activation) may contribute to valvular fibroplasia in humans.

Isoxazolines as potent antagonists of the integrin alpha(v)beta(3).

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.

Disubstituted indazoles as potent antagonists of the integrin alpha(v)beta(3).

A new series of indazole-containing alpha(v)beta(3) integrin antagonists is described. Starting with lead compound 18a, variations in a number of structural features were explored with respect to inhibition of the binding of beta(3)-transfected 293 cells to fibrinogen and to selectivity for alpha(v)beta(3) over GPIIbIIIa, another RGD-binding integrin. Indazoles attached to a 2-aminopyridine or 2-aminoimidazole by a propylene linker at the indazole 1-position and to a diaminopropionate derivative via a 5-carboxylate amide provided the best potency with moderate selectivity. Several differences in the SAR of the diaminopropionate moiety were observed between this series and a series of isoxazoline-based selective GPIIbIIIa antagonists. Compound 34a (SM256) was a potent antagonist of alpha(v)beta(3) (IC(50) 2.3 nM) with 9-fold selectivity over GPIIbIIIa.

alphavbeta3, alphavbeta5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation.

Both smooth muscle cell migration and replication are known to be responsible for neointima formation. Recent reports based on in vitro studies and animal models of neointima formation highlight the possible importance of alphavbeta3 and alphavbeta5 integrins in mediating neointima formation. Clinical data suggest that specific alphavbeta3 blockade may limit restenosis. The aim of this study was to identify the expression of alphavbeta3 and alphavbeta5 and their ligand osteopontin in the very early phases of neointima formation in a rabbit model. A non-occlusive cuff placed around the rabbit femoral artery resulted in a complete, concentric neointima that formed by 14 days. Antibodies specific for the integrin heterodimers and for osteopontin, along with a probe specific for osteopontin mRNA, were used to identify expression at early time points (6 h, 1 day, 3 days, 5 days) post-cuffing. Immunohistochemistry and in situ hybridization expression results were quantitated by image analysis and tested for statistical significance by a two-tailed t-test. The data demonstrated the rapid (within 6 h) and abundant upregulation of alphavbeta3 and alphavbeta5 integrins and their ligand during very early time points of neointima formation. The very early (6 h) upregulation of alphavbeta3 underscores a potentially important clinical intervention point in limiting restenosis following clinical angioplasty procedures.

Disposition and exposure of the fibrinogen receptor antagonist XV459 on alphaIIBbeta3 binding sites in the guinea pig.

The disposition of XV459, a potent, selective GP IIb/IIIa antagonist, has been examined following intravenous administration of XP280, the benzenesulphonate salt, and 3H-SA202, the trifluroacetic acid salt, to male guinea pigs. A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for XV459 quantitation in guinea pig plasma with an LLOQ of 0.1 ng/mL. Intravenous infusions (30 min) of XP280 at doses of 0.5 and 2.0 microg/kg were administered to guinea pigs which were sequentially sacrificed at 0.5, 1, 1.5, 4, 8, 12, 24, 48 and 72 h postinitiation of infusion. Maximum total (unbound and GP IIb/IIIa displaced) XV459 plasma concentration of approximately 3.5 microg/mL was obtained at the 2.0 microg/kg dose. Pooling individual concentration-time data yielded a systemic clearance of 1.42 mL/min/kg, Vss of 0.24 L/kg, and a terminal half-life of 2.8 h in the guinea pig at the 0.5 microg/kg dose. The 2.0 microg/kg dose yielded XV459 exposure that was less than proportional to the previous dose. Similar behaviour has been observed in human trials. Cumulative (up to 72 h) urinary and faecal recovery of total radioactivity was 66.4 and 11.2%, respectively. The time course of spleen, marrow and whole blood radioactivity profiles was similar, suggesting that XV459 was not preferentially sequestered on non-plasma GP IIb/IIIa binding sites. Tissue to blood ratios of 20.7 and 8.3 for the spleen and bone marrow, respectively, indicate that increased (relative to blood) exposure was evident for sites containing the GP IIb/IIIa receptor. In vitro studies confirmed the similarity of XV459 binding to both resting and activated platelets in the guinea pig and humans. Given the comparability of dissociation rate constants and IC50s based on in vitro platelet aggregation, human dosimetry estimates should assume similar partitioning of radiolabelled XV459 as in the guinea pig. These results suggest that the guinea pig may indeed be an appropriate animal model for pharmacokinetic and distribution studies with DMP754; in conjunction with recent pharmacological findings with GP IIb/IIIa antagonists, our results suggest that the guinea pig may be the rodent species of choice for preclinical studies with some other GP IIb/IIIa antagonists.

Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1.

The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.

Antiproliferative gene BTG1 is highly expressed in apoptotic cells in macrophage-rich areas of advanced lesions in Watanabe heritable hyperlipidemic rabbit and human.

In the present study, the expression and potential role of the highly conserved antiproliferative gene BTG1 in the process of apoptosis as it occurs in atherosclerotic lesions was examined. In situ hybridization and immunodetection studies demonstrated that BTG1 localized to specific macrophage-rich regions of lesions in both Watanabe heritable hyperlipidemic rabbits and humans. In addition, the spatial distribution of BTG1 mRNA was shown to colocalize not only with macrophage-rich regions but also to cells that exhibited changes consistent with apoptosis, such as DNA fragmentation and nuclear condensation. In vitro studies demonstrated that forced overexpression of BTG1 induced a 3-fold increase in apoptosis in NIH/3T3 cells. Furthermore, significantly increased expression of BTG1 mRNA resulted from lipid loading of human monocyte-derived macrophages in vitro. The process of increasing lipid content in macrophages is frequently associated with decreased macrophage viability. Taken together, these data underscore a potentially important role for BTG1 in regulating the cellularity of advanced atherosclerotic lesions.

Effects of intracellular free cholesterol accumulation on macrophage viability: a model for foam cell death.

This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-beta-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by electron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor. In addition, inhibition of ACAT for 48 hours resulted in the formation of FC crystals in MPMs but not in J774 cells. If compound 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), which modulates intracellular trafficking of cholesterol, was added together with the ACAT inhibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, because cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabolic changes that were produced when these model foam cells were treated with the ACAT inhibitor parallel the pathological events that have been shown to occur in the developing atherosclerotic plaque.

Bombesin receptor structure and expression in human lung carcinoma cell lines.

Mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory neuropeptides involved in numerous physiologic processes, and have been implicated as autocrine and/or paracrine growth factors in human lung carcinoma. Three structurally and pharmacologically distinct bombesin receptor subtypes have been isolated and characterized: the gastrin releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The three receptors are structurally related, sharing about 50% amino acid identity. They are members of the G-protein coupled receptor superfamily with a seven predicted transmembrane segment topology characteristic of receptors in this family. The signal transduction pathway for GRP-R and NMB-R involves coupling to a pertussis-toxin insensitive G-protein, activation of phospholipase C (PLC), generation of inositol trisphosphate (IP3), release of intracellular calcium, and activation of protein kinase C. While all three bombesin receptors are activated by bombesin agonists, GRP-R, NMB-R, and BRS-3 have very different affinities for the mammalian bombesin-like peptides GRP and NMB, as well as bombesin receptor antagonists. The three bombesin receptor subtypes are expressed in an overlapping subset of human lung carcinoma cell lines. Any therapeutic strategy based on modulation of bombesin growth responses in human lung carcinoma would be well served to take into account the pharmacologic heterogeneity of the relevant receptors.

The ligand binding signatures of the rat AT1A, AT1B and the human AT1 receptors are essentially identical.

The objective of this study was to determine whether the three isoforms of recombinant Ang II receptors (rAT1A, rAT1B and hAT1), stably and individually expressed in CHO cells, could be pharmacologically distinguished by their ligand binding signatures. Competition studies were performed to characterize the inhibition of [125I]Ang II binding to each of the cell membrane preparations by an extensive series of peptide and nonpeptide Ang II analogs. Scatchard plot analyses revealed the following binding characteristics:rAT1A-Kd = 1.27 +/- 0.14 nM; rAT1B- Site 1:Kd = 0.56 +/- 0.11 nM, Site 2: Kd = 126 +/- 23 nM; and hAT1-Site 1:Kd = 1.06 +/- 0.16 nM, Site 2: Kd = 257 +/- 55 nM. The binding of [125I]Ang II in the three preparations was similarly sensitive to inhibition by GTP gamma S. The ligand binding signatures of the three receptor isoforms are essentially the same and are illustrated by the affinity and order of potency of the following ligands: L-158,809 > or = Sar1, Ile8Ang II > saralasin Ang II > or = Ang III > EXP581 > EXP3174 > losartan > or = EXP811 > GR117,289c > EXP6803 > DuP 532 > Ang I >> PD123177. In conclusion, the two rat AT1 receptor isoforms are pharmacologically indistinguishable from each other and from that of the human.

BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells.

The bombesin (BN)-like peptides mediate a diverse spectrum of biological activities and have been implicated as autocrine growth factors in the pathogenesis and progression of some human small cell lung carcinoma tumors. Previously, two mammalian BN-like peptide receptor subtypes, gastrin-releasing peptide receptor and neuromedin-B receptor, have been cloned and characterized. In this study, we have isolated and characterized human genomic and complementary DNA (cDNA) clones encoding a new BN-like peptide receptor subtype, BN receptor subtype 3 (BRS-3). Expression of BRS-3 cDNA in Xenopus oocytes encodes a functional receptor that is specifically activated by BN-like peptides. Chromosome mapping studies indicate that the BRS-3 gene is located on human chromosome X. BRS-3 mRNA expression in rat tissues is limited to secondary spermatocytes in testis. In contrast, BRS-3 mRNA is widely expressed in a panel of human cell lines from all histological types of lung carcinoma. These results suggest a role for BN-like peptides and their receptors in mammalian reproductive physiology and also indicate that BRS-3 could serve as a potential therapeutic target for human lung carcinoma.

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression.

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.

Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

Platelet-derived growth factor-induced destabilization of smooth muscle alpha-actin mRNA.

We have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet-derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha-actin synthesis and SM alpha-actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha-actin mRNA levels. Treatment of postconfluent SMC with both platelet PDGF and recombinant PDGF-BB resulted in a dramatic and concentration-dependent decrease in SM alpha-actin mRNA levels. We observed no differences in efficacy between platelet PDGF and PDGF-BB, indicating that the PDGF-A chain is not required for the effect. The rate of decrease in SM alpha-actin mRNA abundance in PDGF-treated SMC was greater than that observed in cells treated with the transcriptional inhibitor, actinomycin D, with or without PDGF, indicating that PDGF induced a transcriptionally dependent destabilization of the cytosolic SM alpha-actin mRNA pool. This effect appeared selective for SM alpha-actin, in that there was no evidence of a similar change in non-muscle (NM) beta-actin mRNA stability following PDGF treatment. Results of nuclear run-on analyses showed no differences in SM alpha-actin transcription between PDGF- and vehicle-treated SMC at either 4 or 24 hours following treatment, demonstrating that decreases in transcription of the SM alpha-actin gene did not contribute to PDGF-induced changes in SM alpha-actin mRNA abundance. Results of these studies support a possible role for PDGF in regulation of SMC differentiation via a post-transcriptional control mechanism.

RTA, a candidate G protein-coupled receptor: cloning, sequencing, and tissue distribution.

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. Sequence analysis of the genomic clone further showed that the RTA gene has an intron interrupting the region encoding the amino terminus of the protein. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. RTA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. We cannot detect angiotensin binding to the RTA protein after introducing the cognate cDNA or mRNA into COS cells or Xenopus oocytes, respectively, nor can we detect an electrophysiologic response in the oocyte after application of angiotensin peptides. We conclude that RTA is not an angiotensin receptor; to date, we have been unable to identify its ligand.

Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells.

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) alpha-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299-306). Results of this study show that, although there is a 6-fold increase in SM alpha-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM alpha-actin mRNA levels are not different between these cells. This suggests that the SM alpha-actin gene is constitutively active under both of these conditions and that accumulation of SM alpha-actin in postconfluent cells is due to translational and/or post-translational controls. The relationship between growth and cytodifferentiation was further explored by examining the effects of platelet-derived growth factor (PDGF)- or serum-induced growth on actin expression in postconfluent, quiescent cultures maintained in a defined serum-free media. Although both factors have been shown to stimulate proliferation and decrease fractional SM alpha-actin synthesis (Blank et al., 1988), their effects on actin mRNA levels were quite different. PDGF was found to induce a dramatic drop in SM alpha-actin steady state mRNA level but had no effect on nonmuscle beta-actin mRNA level. In contrast, serum stimulation was shown to increase nonmuscle beta-actin mRNA level, whereas SM alpha-actin mRNA level remained constant. Taken together these results indicate that PDGF is a specific and potent repressor of SM alpha-actin expression in vascular SMC and implicate a possible developmental role for PDGF in control of SMC differentiation. In addition, the observation that the level of SM alpha-actin mRNA is unaltered in serum-stimulated cells indicates that an absolute decrease in SM alpha-actin mRNA is not obligatory for cell cycle entrance.