New Author Guidelines for Displaying Data and Reporting Data Analysis and Statistical Methods in Experimental Biology.

The American Society for Pharmacology and Experimental Therapeutics has revised the Instructions to Authors for Drug Metabolism and Disposition, Journal of Pharmacology and Experimental Therapeutics, and Molecular Pharmacology These revisions relate to data analysis (including statistical analysis) and reporting but do not tell investigators how to design and perform their experiments. Their overall focus is on greater granularity in the description of what has been done and found. Key recommendations include the need to differentiate between preplanned, hypothesis-testing, and exploratory experiments or studies; explanations of whether key elements of study design, such as sample size and choice of specific statistical tests, had been specified before any data were obtained or adapted thereafter; and explanations of whether any outliers (data points or entire experiments) were eliminated and when the rules for doing so had been defined. Variability should be described by S.D. or interquartile range, and precision should be described by confidence intervals; S.E. should not be used. P values should be used sparingly; in most cases, reporting differences or ratios (effect sizes) with their confidence intervals will be preferred. Depiction of data in figures should provide as much granularity as possible, e.g., by replacing bar graphs with scatter plots wherever feasible and violin or box-and-whisker plots when not. This editorial explains the revisions and the underlying scientific rationale. We believe that these revised guidelines will lead to a less biased and more transparent reporting of research findings.

New Author Guidelines for Displaying Data and Reporting Data Analysis and Statistical Methods in Experimental Biology.

The American Society for Pharmacology and Experimental Therapeutics has revised the Instructions to Authors for Drug Metabolism and Disposition, Journal of Pharmacology and Experimental Therapeutics, and Molecular Pharmacology These revisions relate to data analysis (including statistical analysis) and reporting but do not tell investigators how to design and perform their experiments. Their overall focus is on greater granularity in the description of what has been done and found. Key recommendations include the need to differentiate between preplanned, hypothesis-testing, and exploratory experiments or studies; explanations of whether key elements of study design, such as sample size and choice of specific statistical tests, had been specified before any data were obtained or adapted thereafter; and explanation of whether any outliers (data points or entire experiments) were eliminated and when the rules for doing so had been defined. Variability should be described by S.D. or interquartile range, and precision should be described by confidence intervals; S.E. should not be used. P values should be used sparingly; in most cases, reporting differences or ratios (effect sizes) with their confidence intervals will be preferred. Depiction of data in figures should provide as much granularity as possible, e.g., by replacing bar graphs with scatter plots wherever feasible and violin or box-and-whisker plots when not. This editorial explains the revisions and the underlying scientific rationale. We believe that these revised guidelines will lead to a less biased and more transparent reporting of research findings.

New Author Guidelines for Displaying Data and Reporting Data Analysis and Statistical Methods in Experimental Biology.

The American Society for Pharmacology and Experimental Therapeutics has revised the Instructions to Authors for Drug Metabolism and Disposition, Journal of Pharmacology and Experimental Therapeutics, and Molecular Pharmacology These revisions relate to data analysis (including statistical analysis) and reporting but do not tell investigators how to design and perform their experiments. Their overall focus is on greater granularity in the description of what has been done and found. Key recommendations include the need to differentiate between preplanned, hypothesis-testing, and exploratory experiments or studies; explanations of whether key elements of study design, such as sample size and choice of specific statistical tests, had been specified before any data were obtained or adapted thereafter; and explanations of whether any outliers (data points or entire experiments) were eliminated and when the rules for doing so had been defined. Variability should be described by S.D. or interquartile range, and precision should be described by confidence intervals; S.E. should not be used. P values should be used sparingly; in most cases, reporting differences or ratios (effect sizes) with their confidence intervals will be preferred. Depiction of data in figures should provide as much granularity as possible, e.g., by replacing bar graphs with scatter plots wherever feasible and violin or box-and-whisker plots when not. This editorial explains the revisions and the underlying scientific rationale. We believe that these revised guidelines will lead to a less biased and more transparent reporting of research findings.

Systematic Fuel Cavity Asymmetries in Directly Driven Inertial Confinement Fusion Implosions.

We present narrow-band self-emission x-ray images from a titanium tracer layer placed at the fuel-shell interface in 60-laser-beam implosion experiments at the OMEGA facility. The images are acquired during deceleration with inferred convergences of ∼9-14. Novel here is that a systematically observed asymmetry of the emission is linked, using full sphere 3D implosion modeling, to performance-limiting low mode asymmetry of the drive.

Late-Time Mixing Sensitivity to Initial Broadband Surface Roughness in High-Energy-Density Shear Layers.

Using a large volume high-energy-density fluid shear experiment (8.5 cm^{3}) at the National Ignition Facility, we have demonstrated for the first time the ability to significantly alter the evolution of a supersonic sheared mixing layer by controlling the initial conditions of that layer. By altering the initial surface roughness of the tracer foil, we demonstrate the ability to transition the shear mixing layer from a highly ordered system of coherent structures to a randomly ordered system with a faster growing mix layer, indicative of strong mixing in the layer at a temperature of several tens of electron volts and at near solid density. Simulations using a turbulent-mix model show good agreement with the experimental results and poor agreement without turbulent mix.

Use of computer vision for analysis of image datasets from high temperature plasma experiments.

Great strides have been made in improving the quality of x-ray radiographs in high energy density plasma experiments, enabled in part by innovations in engineering and manufacturing of integrated circuits and materials. As a consequence, the radiographs of today are filled with a great deal of detail, but few of these features are extracted in a systematic way. Analysis techniques familiar to plasma physicists tend toward brittle 1D lineout or Fourier transform type analyses. The techniques applied to process our data have not kept pace with improvements in the quality of our data. Fortunately, the field of computer vision has a wealth of tools to offer, which have been widely used in industrial imaging and, more recently, adopted in biological imaging. We demonstrate the application of computer vision techniques to the analysis of x-ray radiographs from high energy density plasma experiments, as well as give a brief tutorial on the computer vision techniques themselves. These tools robustly extract 2D contours of shocks, boundaries of inhomogeneities, and secondary flows, thereby allowing for increased automation of analysis, as well as direct and quantitative comparisons with simulations.

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

Observation of persistent species temperature separation in inertial confinement fusion mixtures.

The injection and mixing of contaminant mass into the fuel in inertial confinement fusion (ICF) implosions is a primary factor preventing ignition. ICF experiments have recently achieved an alpha-heating regime, in which fusion self-heating is the dominant source of yield, by reducing the susceptibility of implosions to instabilities that inject this mass. We report the results of unique separated reactants implosion experiments studying pre-mixed contaminant as well as detailed high-resolution three-dimensional simulations that are in good agreement with experiments. At conditions relevant to mixing regions in high-yield implosions, we observe persistent chunks of contaminant that do not achieve thermal equilibrium with the fuel throughout the burn phase. The assumption of thermal equilibrium is made in nearly all computational ICF modeling and methods used to infer levels of contaminant from experiments. We estimate that these methods may underestimate the amount of contaminant by a factor of two or more.

The mating response cascade does not modulate changes in the steady-state level of target mRNAs through control of mRNA stability.

Many extracellular signals trigger changes in gene expression by altering the steady-state level of target transcripts. This modulation of transcript levels is typically ascribed to changes in transcription of target genes; however, there are numerous examples of changes in mRNA processing and stability that contribute to the overall change in transcript levels following signalling pathway activation. The alpha-factor-stimulated mating pathway in Saccharomyces cerevisiae is a receptor-operated MAP kinase cascade that results in increased levels of a large number of target mRNA transcripts when stimulated acutely. A previous study identified many of the transcripts modulated in response to alpha-factor and argued, based on genetic studies, that the response occurred solely at the level of gene transcription (Roberts et al., 2000). We directly examined whether enhanced mRNA stability contributes to the increase in the steady-state level of alpha-factor target transcripts by exploiting a temperature-sensitive RNA Polymerase II mutant, a Ste12 transcription factor import mutant, and tet-regulated synthetic mating factor minigene reporters. Examination of a panel of alpha-factor-responsive transcripts reveals no change in mRNA stability in response to alpha-factor stimulation, providing direct evidence that this signal transduction pathway in S. cerevisiae does not function through modulating transcript stability.

Homeostatic Intrinsic Plasticity Is Functionally Altered in Fmr1 KO Cortical Neurons.

Cortical hyperexcitability is a hallmark of fragile X syndrome (FXS). In the Fmr1 knockout (KO) mouse model of FXS, cortical hyperexcitability is linked to sensory hypersensitivity and seizure susceptibility. It remains unclear why homeostatic mechanisms fail to prevent such activity. Homeostatic intrinsic plasticity (HIP) adjusts membrane excitability through regulation of ion channels to maintain activity levels following activity perturbation. Despite the critical role of HIP in the maturation of excitability, it has not been examined in FXS. Here, we demonstrate that HIP does not operate normally in a disease model, FXS. HIP was either lost or exaggerated in two distinct neuronal populations from Fmr1 KO cortical cultures. In addition, we have identified a mechanism for homeostatic intrinsic plasticity. Compromising HIP function during development could leave cortical neurons in the FXS nervous system vulnerable to hyperexcitability.

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.

Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.

Activation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle cells.

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT-mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT-responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

Ubiquitin-proteasome degradation of KLF5 transcription factor in cancer and untransformed epithelial cells.

Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules such as p53, cyclins, myc, BRCA1, HIF-1alpha, etc. The Kruppel-like factor 5 (KLF5) transcription factor regulates biological processes involved in carcinogenesis, angiogenesis, and smooth muscle cell differentiation. In carcinogenesis, KLF5's role has been indicated by frequent genetic deletion as well as functional studies. Here we show that KLF5 is an unstable protein with a short half-life. Destruction of KLF5 was prevented by each of the proteasome-specific inhibitors tested but not by an inhibitor for trypsin-like proteases and cysteine proteases or by a lysosome inhibitor in epithelial cells. Furthermore, KLF5 underwent ubiquitination, and deletion of a 56-amino-acid sequence adjacent to a known transactivation domain of KLF5 significantly reduced its ubiquitination and degradation. Interestingly, cancer cells appeared to be more active in KLF5 degradation than untransformed epithelial cells, yet their proteasome activity was not higher. These results suggest that KLF5 protein is degraded at least in part through ubiquitination-proteasome pathway, which may have become hyperactive for KLF5 in cancer cells.

Organization of the bovine gene encoding the endothelial nitric oxide synthase.

The bovine endothelial nitric oxide synthase gene plus 2.9 kilobases of 5'-flanking sequence has been isolated and characterized. The gene spans 20 kilobases and contains 26 exons and 25 introns. Two transcription start sites have been determined by primer extension analysis which are located 170 and 240 base pairs upstream, respectively, from the methionine translational initiation codon. Evidence supporting the upstream boundary region for transcriptional initiation was also obtained by reverse transcription-polymerase chain reaction. The 5'-flanking region lacks a typical TATA box but contains numerous putative transcription factor binding sites. These include consensus sequences for an AP-1 site, an NF-1 site, a tumor necrosis factor responsive element, two sterol regulatory elements, 3 acute-phase response element, two sterol regulatory elements, 3 acute-phase response elements, 6 GATA motifs, 16 CACCC boxes, 5 Sp1 sites, 15 estrogen half-palindromic motifs, and 9 fluid shear stress-responsive elements. The isolated gene promoter directs basal transcription of a luciferase reporter gene when transiently transfected into bovine aortic endothelial cells. High sequence homology of the promoter region to the human endothelial nitric oxide synthase gene promoter (75% nucleotide identity in 1.6 kilobases of 5'-flanking sequence) suggests evolutionary conservation of transcriptional regulation. Isolation and characterization of the bovine endothelial nitric oxide synthase gene should facilitate further investigation of mechanisms by which gene expression is regulated.

Difficulty of extraction of chronically implanted tined ventricular endocardial leads.

The dislodgment rate of permanent pacing ventricular and atrial endocardial leads has significantly decreased with the incorporation of tines as a fixation device. In contrast, transvenous manual extraction of chronically implanted endocardial leads is, at times, clinically indicated, particularly when pacemaker system infection is present. The success rate of such extraction attempts for ventricular endocardial leads over the past 5 years was reviewed. Extraction was usually successful (six of seven attempts) in patients with silicone rubber nontined (or short-tined) older ventricular endocardial leads (Group A). However, in patients with newer urethane long-tined ventricular endocardial leads (Group B), extraction was unsuccessful in three of four attempts. Because of entrapment of the distal electrode tip in the right ventricular apex, manual traction of these leads resulted in permanent conductor material stretching with resultant urethane insulator material breakage in the region of the joints with proximal and distal electrodes. The one successful extraction in Group B was technically difficult and appeared to create a significant risk of intracardiac lead separation. This experience indicates that with improved pacemaker lead design decreased lead dislodgment has been obtained at the cost of increased difficulty of ventricular endocardial lead extraction. Such difficulty should be anticipated when a clinical decision is made to attempt to extract the new urethane long-tined ventricular leads.

Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal.

Functional expression of the transcriptional activator Runx2/Cbfal is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.

Carotid sinus hypersensitivity: beneficial effects of dual-chamber pacing.

Three types of carotid sinus (CS) syndrome have been described: cardioinhibitory, vasodepressor and mixed. For the treatment of symptomatic patients with associated significant cardioinhibition, permanent ventricular demand pacing systems are often implanted. Even with this pacing modality, some patients remain symptomatic because of continued (and at times aggravated) vasodepression. This study assesses the effects of loss of atrial preloading and orthostasis after carotid massage in patients with CS hypersensitivity. Eleven patients were studied using constant intra-arterial pressure measurements during either ventricular (VVI) or atrioventricular sequential (DVI) pacing in both supine or upright positions. The measurements performed included the magnitude of decrease in arterial blood pressure (BP), the rate of decrease of BP and the percent change in BP from baseline values. After carotid massage, all 11 patients had greater hemodynamic change with the VVI than DVI pacing mode, whether in the supine or upright position. The decreases in systolic BP were: DVI (supine) 29 mm Hg, VVI (supine) 48 mm Hg, DVI (upright) 37 mm Hg, and VVI (upright) 59 mm Hg (mean group values, p less than 0.001). The rates of decrease of systolic BP were: DVI (supine) 2.9 mm Hg/s, VVI (supine) 5.7 mm Hg/s, DVI (upright) 4.1 mm Hg/s, and VVI (upright) 8.3 mm Hg/s (mean group values, p less than 0.001). VVI pacing, particularly in the upright position, resulted in a significant increase in the incidence of patient symptoms (p = 0.03). Thus, in CS hypersensitivity, VVI pacing results in significant hemodynamic deterioration compared to DVI mode. This aggravation of the vasodepressor component results in increased patient symptoms, and therefore, DVI is the optimal pacing mode.

Neurogenic plasma leakage in mouse airways.

1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature.

Molecular cloning of AT1 angiotensin receptors.

Angiotensin II is the principal effector molecule of the renin-angiotensin system. Its effects are mediated by cell surface proteins termed AT receptors. On the basis of radioligand binding studies, these have been pharmacologically subdivided into two classes, termed AT1 receptors and AT2 binding sites (Chiu AT, et al, Biochem Biophys Res Commun 1989;165:196-203). AT1 receptors appear to mediate the major cardiovascular effects of angiotensin II, whereas no known physiological properties appear to be coupled to AT2 binding sites (Wong PC, et al, J Pharmacol Exp Ther 1990;255:584-592). To gain further insight into the function of AT1 receptors we have isolated rat cDNA's and genes encoding two distinct but highly similar isoforms of AT1 receptors, termed AT1a and AT1b receptors. Two cDNA's encoding the vascular AT1a receptor were isolated by an expression cloning strategy from a cDNA library prepared from vascular smooth muscle cells. The properties of the clones isolated by this approach are consistent with known pharmacological, biochemical signaling, and tissue distribution properties of AT1 receptors. Using this cDNA as a probe, a second isoform of rat AT1 receptor was isolated from a genomic library. This receptor, termed the AT1b receptor, is 95% identical in amino acid sequence and is pharmacologically indistinguishable from the AT1a receptor. However, the tissue-specific expression pattern of the AT1b gene differs significantly from that for the AT1a receptor.