Molecular genetic analysis of 30 families with Joubert syndrome.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Measurement and compartmental modeling of the effect of CYP3A5 gene variation on systemic and intrarenal tacrolimus disposition.

We evaluated the hypothesis that cytochrome P450 3A5 (CYP3A5) expression can affect intrarenal tacrolimus accumulation. Tacrolimus was administered orally to 24 healthy volunteers who were selected on the basis of their CYP3A5 genotype. As compared with CYP3A5 nonexpressors, expressors had a 1.6-fold higher oral tacrolimus clearance and 2.0- to 2.7-fold higher metabolite/parent area under the curve (AUC) ratios for 31-desmethyl tacrolimus (31-DMT), 12-hydroxy tacrolimus, and 13-desmethyl tacrolimus (13-DMT). In addition, the apparent urinary tacrolimus clearance was 36% lower in CYP3A5 expressors as compared with nonexpressors. To explore the mechanism behind this observation, we developed a semiphysiological model of renal tacrolimus disposition and predicted that tacrolimus exposure in the renal epithelium of CYP3A5 expressors is 53% of that for CYP3A5 nonexpressors, when normalized to blood AUC. These data suggest that, at steady state, intrarenal accumulation of tacrolimus and its primary metabolites will depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient differences in the risk of tacrolimus-induced nephrotoxicity.

The clinical significance of Cyclin B1 and Wee1 expression in non-small-cell lung cancer.

BACKGROUND: Cyclin B1 has an important role in the G2-M phase transition of the cell cycle. Wee1 delays mitosis by suppressing the activity of the Cyclin B1/cdc2 complex. The objective of the present study was to elucidate the clinicopathological and prognostic significance of Cyclin B1 and Wee1 expression in non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: An immunohistochemical assessment of Cyclin B1 and Wee1 expression was performed in 79 patients with NSCLC. RESULTS: The expression of Cyclin B1 was correlated with differentiation (P = 0.0423) and vascular invasion (P = 0.001). Patients with overexpression of Cyclin B1 had higher mean values for both the Ki-67 proliferative index (Ki-Index) (P <0.0001) and proliferating cell nuclear antigen labeling index (PCNA-LI) (P <0.0001), and a poorer prognosis (P = 0.0068). Patients lacking expression of Wee1 had a higher recurrence rate (P = 0.0084) and a poorer prognosis (P = 0.0457), and tended to have higher Ki-Index and PCNA-LI values. Multivariate analysis suggested that both Cyclin B1 (P = 0.0244) and Wee1 (P = 0.0444) expression were significant prognostic factors. CONCLUSIONS: These findings suggest that Cyclin B1 expression could be a significant prognostic parameter in NSCLC. The loss of Wee1 expression may have a potential role in promoting tumor progression and may be a significant prognostic indicator in NSCLC.

Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from the brain across the blood-brain barrier.

The contribution of organic anion transporters to the total efflux of 17beta-estradiol-D-17beta-glucuronide (E(2)17betaG) through the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method by examining the inhibitory effects of probenecid, taurocholate (TCA), p-aminohippurate (PAH), and digoxin. E(2)17betaG was eliminated through the BBB with a rate constant of 0.037 min(-1) after the microinjection into the brain. Probenecid and TCA inhibited this elimination with an IC50 value of 34 and 1.8 nmol/0.5 microl of injectate, respectively, whereas PAH and digoxin reduced the total efflux to about 80 and 60% of the control value, respectively. The selectivity of these inhibitors was confirmed by examining their inhibitory effects on the transport via organic anion transporting polypeptide 1 (Oatp1), Oatp2, organic anion transporter 1 (Oat1), and Oat3 transfectants using LLC-PK1 cells as hosts. Digoxin specifically inhibited the transport via Oatp2 (K(i) = 0.037 microM). The K(i) values of TCA for Oatp1 and Oatp2 (11 and 39 microM, respectively) were about 20 times lower than those for Oat1 and Oat3 (2.8 and 0.8 mM, respectively). PAH did not affect the transport via the Oatp family, but had a similar affinity for Oat1 and Oat3 (85 and 300 microM, respectively). Probenecid had a similar affinity for these transporters (Oatp1, Oatp2, Oat1, and Oat3) examined in this study. Taking the selectivity of these inhibitors into consideration, the maximum contribution made by the Oatp2 and Oat family to the total efflux of E(2)17betaG from the brain appears to be about 40 and 20%, respectively.

Oral absorption of cephalosporins is quantitatively predicted from in vitro uptake into intestinal brush border membrane vesicles.

In order to establish a method to predict oral absorption of drugs, which are absorbed by the oligopeptide transporter (PepT1), fraction absorbed (F) of cephalosporin antibiotics was predicted from in vitro uptake into rat intestinal brush border membrane vesicles (BBMV). Using in vitro uptake data, F values of cephalosporins in humans were predicted using the equation derived from the complete radial mixing (CRM) model, which was proposed by Amidon et al. (Amidon et al., J. Pharm. Sci. 69 (1980) 1369). In the present study, uptake into BBMV was measured at 25 and 4 degrees C in the presence of an H+ -gradient, and the uptake clearance (CLuptake) was calculated. Clearance for the uptake mediated by PepT1 (DeltaCLuptake) was then calculated as CLuptake at 25 degrees C minus that at 4 degrees C. When DeltaCLuptake and F values were analyzed according to the present equation, fairly good correlation between DeltaCLuptake and F was observed. It was further demonstrated that the present method may be able to quantitatively predict F values of cephalosporins by using several cephalosporins as standards.

An easy method for the intraluminal administration of peppermint oil before colonoscopy and its effectiveness in reducing colonic spasm.

BACKGROUND: Systemic administration of a cholinergic blocking agent or glucagon is used to reduce spasms, but it is inconvenient and sometimes causes side effects. This study is an evaluation of the intracolonic administration of peppermint oil during colonoscopy for the control of colonic spasm. METHODS: Each patient in the treated group (n = 409) was given approximately 200 mL of the solution (a mixture of 8 mL of peppermint oil and 0.2 mL of Tween 80 per 1 L of water with 0.04% indigo carmine) by using a hand pump attached to the accessory channel of the colonoscope. Changes in patient posture were made to distribute the solution. The patients in the control group (n = 36) were given the solution without peppermint oil. RESULTS: A satisfactory spasmolytic effect was seen in 88.5% of the treated patients and in 33.3% of those in the control group (p<0.0001). No adverse effect was observed. The mean time to onset was 21.6 +/- 15.0 seconds, and the effect continued for at least 20 minutes. In patients with irritable bowel syndrome, efficacy was significantly lower (p < 0.0001). CONCLUSIONS: The intraluminal administration of peppermint oil by using a hand pump is a simple, safe, and convenient alternative to the systemic injection of a cholinergic blocking agent or glucagon during colonoscopy.

Invasive pulmonary aspergillosis in a puerperant with drug-induced agranulocytosis.

Invasive pulmonary aspergillosis (IPA) is an acute infection of Aspergillus species to the lungs. It generally occurs in immunocompromised hosts, especially with neutropenia. We report a 30-year-old puerperant, who developed IPA from agranulocytosis. She had been treated for threatened labor with ritodrine and cefepime, one of which induced agranulocytosis. After vaginal delivery of twins, pneumonia emerged in the right lower lobe. She was diagnosed to have IPA according to the halo sign on computed tomography (CT) and positive circulating antibody against Aspergillus, and was treated successfully with oral itraconazole followed by surgical resection. It is important to note that IPA might arise in otherwise immunocompetent hosts when neutropenia is long-standing.

Molecular analysis of the h-warts/LATS1 gene in human breast cancer.

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.

Loss of standard type of CD44 expression in invaded area as a good indicator of lymph-node metastasis in colorectal carcinoma.

PURPOSE: Recent advances have made possible the treatment of small invasive colorectal cancer by means of polypectomy or endoscopic mucosal resection. CD44 expression in cancer cells was identified as an indicator of lymph-node metastasis, which could be evaluated in specimens removed by colonoscopy. METHODS: The correlation between lymph-node metastasis and the expression of standard-type CD44 in cancer cells was examined immunohistologically using the invaded cancer cells of 61 tissue samples of superficially invasive colorectal cancer. We defined the above as invasive cancer restricted within the colorectal wall. Of the 61 samples, 31 had submucosal invasion and 30 had muscular invasion. RESULTS: Standard-type CD44 expression in the area of invasion in cases with lymph-node metastasis was remarkably down-regulated. In 43 cases with no lymph-node metastasis, 36 (83.7 percent) of patients had CD44 expression in invaded cells, whereas only two of 18 cases (11.1 percent) with lymph-node metastasis had expression of standard-type CD44 in the same area (P < 0.0001). A total of 69.6 percent (16/23) of patients with loss of standard-type CD44 expression in invaded sites were found to have positive metastasis in the lymph nodes. These results suggest that standard-type CD44 in invasive colon cancer cells could suppress metastasis to the regional lymph nodes. CONCLUSION: In cases of invasive colorectal cancer, the loss of standard-type CD44 expression in the invaded area is a sensitive marker for metastasis to the lymph nodes. Further investigation with larger patient groups is required to clarify the reliability of loss of standard-type CD44 expression as an indicator for additional surgery after endoscopic resection of submucosal invasive colorectal carcinoma.

Mutation analysis of transforming growth factor beta type II receptor, Smad2, Smad3 and Smad4 in esophageal squamous cell carcinoma.

Mutations of the transforming growth factor beta type II receptor (TGFbetaRII) gene and Smad family genes have been observed in several human cancers. However, there has been no report on mutation analysis of the entire coding regions of these genes in esophageal cancer, and the role of these genes in the development of esophageal cancer remains unknown. We performed polymerase chain reaction-single strand conformation polymorphism analysis of TGFbetaRII, Smad2, Smad3 and Smad4 genes and microsatellite assay in 20 esophageal squamous cell carcinomas (ESCC). We detected polymorphisms at exon 2 of Smad3 and intron 6 of Smad4. No mutation was found in TGFbetaRII, Smad2, Smad3 and Smad4 genes. These results suggest that mutation of TGFbetaRII, Smad2, Smad3 and Smad4 genes is a rare event in ESCC.

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma.

BACKGROUND: KAI1/CD82, a tumor metastasis suppressor gene, is correlated inversely with the progression and invasion of several tumors. It also has been reported that the KAI1 gene is related to the tumor suppressor gene p53. This study was performed to clarify the correlation between KAI1/CD82 expression and clinicopathologic characteristics and p53 expression in patients with esophageal squamous cell carcinoma (ESCC). The authors also investigated mutation of the KAI1 gene coding region to determine whether this may reduce KAI1 expression in ESCC. METHODS: Using immunohistochemistry with anti-KAI1 polyclonal antibody and monoclonal antibody against p53, KAI1/CD82 and p53 expression were detected in 55 patients with ESCC who had undergone surgery. The authors examined the KAI1 gene mutation in 22 patients with ESCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing. RESULTS: KAI1/CD82 expression was positive in 36 of 55 patients (65.5%). There was a significant inverse correlation between KAI1/CD82 expression and regional lymph node metastasis (P = 0.0045), distant metastasis (P = 0.0092), the number of lymph node metastases (P = 0.0019), and pathologic stage (P = 0.0046). The survival rates of KAI1/CD82 negative patients were poorer than those of positive patients (P = 0. 024). The correlation between KAI1 positive and p53 positive tumors was not statistically significant. None of the 22 patients with ESCC showed mutation of the KAI1 gene by PCR-SSCP. In one patient, there was polymorphism in the SSCP assay and DNA sequencing. CONCLUSIONS: The authors demonstrated immunohistochemically that the expression of KAI1 protein appeared to be correlated with lymph node metastasis. Mutation does not seem to be a mechanism for dysregulation of the KAI1 protein in ESCC.

Genetic variations of the midkine (MK) gene in human sporadic colorectal and gastric cancers.

Midkine (MK), a retinoic acid responsible protein, is regulated during development and may play an important role in tumorigenesis. A search for genetic variations of the MK gene, located on chromosome 11q11.2 in humans, has not yet been conducted in cancers. To examine the entire coding region, as well as 4 regions of the promoter covering all functional motifs, 8 sets of intron-based and promoter region primers were designed. Using these primers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of genomic DNA samples from 60 sporadic colorectal and 37 sporadic gastric cancer patients was carried out. This analysis, followed by DNA sequencing, revealed a heterozygous g/t polymorphism at the 62nd base on intron 3 in five colorectal tumors (8.3%) and one gastric tumor (2.7%). In the promoter region, a heterozygous CTT deletion, creating a (CTTTT)2 repeat, in one colorectal cancer sample (1.67%) and a heterozygous 2-bp deletion in the G7 tract in another colorectal cancer patient were detected. A/G and A/A alleles were also detected at nt. -1741 in 36 (97.3%) and one (2.7%) gastric cancer samples, respectively. The A/G alleles were observed in all colorectal cancer patients (100%). All variations observed in the promoter region showed polymorphism. These results suggest that in sporadic colorectal and gastric cancers some gene alterations are present in the MK promoter region, but alterations in the coding region are rare.

Mutation analysis of the Smad3 gene in human ovarian cancers.

The Smad3 gene is a member of the Smad family, vertebrate homologues of Drosophila Mad, and its gene product is a cytoplasmic element in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad3 gene mutations in 36 human ovarian cancers, and found that 15 cases (41. 7%) had a polymorphism at codon 103. Because this mutation was not accompanied by amino acid replacement, the present results show that the mutations in the Smad3 gene are unlikely to be involved in human ovarian cancers.

Alterations of p73 preferentially occur in gastric adenocarcinomas with foveolar epithelial phenotype.

To establish the possible involvement of p73, a newly discovered p53-related candidate as a tumor-suppressor gene in human stomach carcinogenesis, the allelic status, allele-specific expression and mutations of the gene were investigated using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, RT-PCR SSCP analysis and direct DNA sequencing in 95 gastric adenocarcinomas. Of these, 32 exhibited the heterozygous p73 allele for the StyI restriction site in exon 2. Among these, the cancer DNA of 12 revealed loss of heterozygosity (LOH) of p73. All of the cancers with p73 LOH exhibited phenotypes of foveolar epithelium of the stomach. RT-PCR SSCP analysis of p73 heterozygous cases demonstrated not only bi-allelic expression of the gene but also relatively reduced expression of the affected allele in 6 of 8 tumors with p73 LOH. No gene mutation was detected in the remaining allele of LOH-positive cancers. Our results suggest that alterations of p73, including LOH and abnormal expression, may play roles in the genesis of foveolar-type gastric adenocarcinomas, though this is not in line with a classical Knudson's "2-hit" model.

No mutations of the Smad2 gene in human sporadic gastric carcinomas.

BACKGROUND: The majority of cancer cells escape from TGF-beta-mediated growth control. However, the mechanism of resistance to the growth inhibitory effects by TGF-beta is not clear. TGF-beta signaling is initiated when the type I receptor phosphorylates the SMAD proteins, Smad2 and Smad3. Recently, mutations of Smad2 have been detected in human colon and lung cancers. Mutation of coding sequences of Smad2 in gastric carcinomas has not yet been elucidated adequately. METHODS: PCR-SSCP analysis of the entire coding region of Smad2 in 35 human sporadic gastric cancers and eight gastric cancer cell lines was performed using 11 sets of intron-based primers. RESULTS: No mutations of Smad2 were detected in any tumor or cell line. CONCLUSIONS: The results suggest that mutation of Smad2 does not play a key role in human stomach carcinogenesis.

Effect of brefeldin A and lysosomotropic reagents on intracellular trafficking of epidermal growth factor and transferrin in Madin-Darby canine kidney epithelial cells.

To regulate intracellular sorting of epidermal growth factor (EGF) or transferrin (Tf), the effect of brefeldin A (BFA) or lysosomotropic reagents was investigated. To examine the effect of them on the net transcellular transport of 125I-EGF or 125I-Tf, their transcytosis was investigated in the presence or absence of reagents. For the investigation of their fate after internalization, radiolabeled ligands were internalized at 37 degreesC, followed by extensive washing and subsequent incubation at 37 degreesC in the ligand-free medium (pulse-chase study). BFA enhanced transcytosis of 125I-Tf, but had no effect on 125I-EGF. Kinetic analysis in the pulse-chase study showed that BFA does not affect cell-surface binding or intracellular sorting of EGF, while it only increases the transcytosis rate constant of Tf. From the lysosomotropic reagents study, both ammonium chloride and monensin suppressed transcytosis and recycling as well as the degradation of EGF, while both chloroquine and bafilomycin A selectively suppressed the degradation process with only a minimal effect on transcytosis, resulting in an increase in the amount transcytosed. It is concluded the that enhancement effect of BFA on transcytosis depends upon the type of receptor targeted. Lysosomotropic reagents can be divided into two types as far as the specificity of the effect on the net amount of EGF transcytosed in Madin-Darby canine kidney (MDCK) cells is concerned.

Mutation of the transforming growth factor-beta type II receptor gene is a rare event in human sporadic gastric carcinomas.

Mutations of the transforming growth factor-beta type II receptor (TGF-beta RII) gene have been detected in several human cancers. However, mutation analysis of coding sequences of TGF-beta RII in gastric carcinomas has not yet been fully elucidated. We performed PCR-SSCP analysis and direct DNA sequencing of the entire coding region of TGF- RII in 38 human sporadic gastric cancers and 8 gastric cancer cell lines. Mutations of the TGF-beta RII were detected in two tumors and three cell lines. Two tumors had one base deletion in the polyadenine tract in exon 3, the cystein-rich extracellular domain. Three cell lines had a silent mutation in the kinase domain located in exon 4. Polymorphisms were detected in introns 2 and 3. An a/g polymorphism was observed at the seventh base in intron 2 and an a/t polymorphism was observed at the fourth to last base in intron 3. There were no mutations in exons 1, 2, 5, 6 and 7. These results indicate that the polyadenine tract in the TGF-beta RII is a mutational hot spot in human gastric cancer. However, these results also suggest that mutations of the gene are rare events in human sporadic gastric cancer.

Kinetic analysis of transcytosis of epidermal growth factor in Madin-Darby canine kidney epithelial cells.

PURPOSE: The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells. METHODS: The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface. 125I-EGF was prebound to the basal surface at 4 degrees C, followed by extensive washing and subsequent incubation at 37 degrees C in EGF-free medium. RESULTS: Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (approximately 90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1-3 min, followed by intracellular degradation with a half-life of 20-50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130-250 min. Even when 125I-EGF was incubated with MDCK cells at 37 degrees C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160-170 min. CONCLUSIONS: The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.

The endothelin receptor is a major determinant for the nonlinear tissue distribution of the endothelin antagonist BQ-123.

The nonlinearity in the pharmacokinetics of the cyclopentapeptide endothelin antagonist BQ-123 was studied. Both the total body clearance and tissue-to-plasma concentration ratio (Kp) were investigated in rats under a wide range of steady-state plasma concentrations (Cpss) obtained by changing the intravenous infusion rate of BQ-123. The total body clearance was constant up to a Cpss level of 50 microM, although it was markedly decreased at higher Cpss values, which suggests the existence of a saturable elimination mechanism. A Cpss-dependent nonlinearity in the apparent Kp values (Kp,app) was clearly observed in many organs including lung, heart, spleen, pancreas, adrenal, stomach, intestine, colon, aorta, testis and muscle, where the endothelin ET(A) receptor is known to be localized. By fitting the saturation curves of the Kp,app values, a similar dissociation constant (Kd) was obtained for most organs at 5 to 10 nM, which is close to the reported Kd values of BQ-123 for the endothelin ET(A) receptor. The saturable portion of the Kp,app values observed in vivo showed a good correlation with reported values of the endothelin ET(A) receptor density. Binding of BQ-123 to isolated membrane fractions from several organs demonstrated clear saturability for the lung, heart, spleen and liver with Kd values of 1 to 3 nM. Such specific binding also showed a good correlation with the saturable portion of the Kp,app values. From these results, we concluded that the endothelin receptor(s) is responsible for the nonlinear tissue distribution of BQ-123 in rats.