Biomedical subjects

H L Wu

Publications and source records attributed to H L Wu.

At least 73 records · Page 4Linked to original sources

PubMed1997

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene.

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed W138 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and W138 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed W138 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylated RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.

3T3 Cells↗

PubMed1997

Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity.

Both mice and rabbits immunized with dengue virus E protein peptide spanning amino acids 100-119 (D4E) produced antibodies that reacted not only with the D4E peptide itself but also with human plasminogen, as shown by ELISA and Western blot. Sera from dengue virus-hyperimmunized mice and dengue patients also contained antibodies against D4E and plasminogen. Furthermore, such sera all contained plasmin inhibitory activity. Using affinity-purified anti-D4E antibodies and free D4E peptide for competitive inhibition, we demonstrated that the inhibition of plasmin activity was due to anti-D4E antibodies rather than other substances in the sera. Taken together, these results suggest dengue virus E protein amino acids 100-119 are a cross-reactive immunogenic region, and antibodies against this region may interfere with human fibrinolysis.

Animals↗

PubMed1997

Susceptibility of three laboratory strains of Aedes albopictus (Diptera: Culicidae) to Japanese encephalitis virus from Taiwan.

The susceptibility of 3 laboratory strains of Aedes albopictus (Skuse) (Sanhsia [SH], Yungho [YH], Liyang [LY], and 1 strain of Culex tritaeniorhynchus Giles from northern and central Taiwan were compared for susceptibility to the MQ1-2 strain of Japanese encephalitis (JE) virus. The median infective dose (MID50) by intrathoracic inoculation was 0.23, 0.76, 1.60, and -0.03 log10 WMICLD50 (50% weanling mice intracranial lethal dose) with Ae. albopictus SH, YH, LY, and Cx. tritaeniorhynchus, respectively. After feeding on a sweetened blood-virus mixture, the oral MID50 was 2.03, 4.32, and 4.98 log10 WMICLD50 for SH, YH, and LY, respectively, and 1.02 log10 WMICLD50 for Cx. tritaeniorhynchus. The SH Ae. albopictus strain transmitted virus to normal mice after 14 d. with an average transmission rate of 45%. Based on these results, the SH strain was the most susceptible and important potential vector among 3 Ae. albopictus strains for the sympatric MQ1-2 strain of JE.

Aedes↗

PubMed1997

Molecular biology of hepatitis D virus: research and potential for application.

Superinfection by hepatitis D virus (HDV) leads to acute hepatitis and causes progression to liver cirrhosis in a significant proportion of hepatitis B surface antigen (HBsAg) carriers. Current regimens (interferon) to treat hepatitis D patients has only transient but no lasting effects. New approaches are, therefore, warranted. Recently, several laboratory studies have discovered interesting properties of HDV that may become targets for antiviral chemicals. Viral replication requires the small hepatitis delta antigen (s-HDAg). The s-HDAg is a nuclear phosphoprotein. There is evidence indicating that phosphorylation is important for HDV replication. A second step of replication requires HDV-RNA self-cleavage and self-ligation. Interestingly, one group of antibiotics, the aminoglycosides, exerts strong suppression effects on HDV ribozyme activities. In the following stage of viral assembly, two post-translational modifications, namely isoprenylation of large HDAg and glycosylation of HBsAg are involved. Agents capable of blocking the two modifications should reduce viral production. These four possible targets are reviewed. For prevention, effective vaccines are not yet available. Two novel approaches are discussed. The first demonstrates the immunogenicity of a nucleic acid vaccine in mice. The second approach assembled an empty HDV particle in yeast. Advances on such laboratory investigations may provide new methods for the control of hepatitis D in the future.

Aminoglycosides↗

PubMed1997

[Comparative study on the chimeric ability of embryonic stem cell lines HDC and MESPU-13].

While producing transgenic mice by ES cell route, it is important to know whether ES cells used have a strong ability to produce chimeras or not. In this report, two methods were used for studying the chimeric ability of two ES cell lines (HDC and MESPU-13). One method was the blastocyst microinjection, that is, chimeras were produced by injecting 15 ES cells into the cavity of C57BL/6J blastocysts. Another was GPI electrophoresis for examinizing the chimerism of ES cells in internal tisssues and organs of chimeras. The results showed that the ability of producing chimeras of MESPU-13 cells was stronger than that of HDC cells. The reason was discussed in detail.

Animals↗

PubMed1996

Molecular characterization of a novel human endothelin receptor splice variant.

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

Alternative Splicing↗

PubMed1996

Regulation of plasminogen activator inhibitor activity by plasmin in endothelial cells.

The fibrinolytic activity in endothelial cells was regulated by balance of plasminogen activators and plasminogen activator inhibitors. Plasmin can specifically inhibit the biosynthesis of tissue-type plasminogen activator (t-PA), but not plasminogen activator inhibitor, type 1 (PAI-1) in endothelial cells. The PAI activity in the conditioned medium of endothelial cells was low and remained constant in 24 hours. However, the PAI activity in the conditioned medium of the plasmin-pretreated cells increased linearly in 24 hours. Pretreatment with protein kinase C inhibitors, H-7 or staurosporine, partially suppressed the PAI activity induced by plasmin. Pretreatment of endothelial cells with a G-protein inhibitor pertussis toxin resulted in an inhibition of the plasmin-induced PAI activity. The phospholipase A2 inhibitor mepacrine specifically eliminated the effect of plasmin stimulation on PAI activity. Cyclooxygenase and lipoxygenase inhibitors also partially inhibited the plasmin-stimulated PAI activity in endothelial cells. All these inhibitors did not affect the biosynthesis of the PAI-1 antigen in the presence or absence of plasmin. The results indicate that plasmin increased the PAI activity of endothelial cells via pathways in which protein kinase C, G protein, and phospholipase A2 may be involved.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Hepatitis C virus RNA in peripheral blood mononuclear cells: comparing acute and chronic hepatitis C virus infection.

Hepatitis C virus (HCV) can infect peripheral blood mononuclear cells (PBMC) of patients with chronic HCV infection. No data are available on PBMC testing for HCV RNA in acute hepatitis C. This study investigated the presence of HCV RNA in PBMC of patients with acute posttransfusion hepatitis C, compared with those with chronic HCV infection. Nested polymerase chain reaction (PCR) was applied to detect HCV RNA in 111 and 48 paired samples of serum and PBMC of 11 patients with acute posttransfusion hepatitis C and 48 patients with chronic HCV infection, respectively. In patients with acute posttransfusion hepatitis C, HCV RNA was detected in 17 of 29 (59%) and 67 of 82 (82%) serum samples collected during the incubation period and acute phase, respectively. Meanwhile, of the 48 patients with chronic HCV infection, 41 had serum HCV RNA (85%). HCV RNA was not detected in PBMC samples from incubation period or from acute-phase hepatitis, although it was detected in 12 of the 48 PBMC samples of chronically infected patients (25%) P < .005). Of the 12 PBMC specimens positive for positive-stranded HCV RNA, 6 were also positive for negative-stranded HCV RNA. Among patients with chronic HCV infection, HCV infection of PBMC was not related to age, sex, blood transfusion, serum alanine transaminase (ALT) levels, or serum virus titers. In conclusion, HCV infection of PBMC rarely exists in patients with acute hepatitis C. As HCV infection persists, the incidence of HCV infection of PBMC becomes higher.

Acute Disease↗

PubMed1996

Incidence of post-transfusion hepatitis in Taiwan before and after introduction of anti-HCV testing.

The effectiveness of second-generation anti-hepatitis C virus antibody (anti-HCV) screening of blood donations for the prevention of non-A, non-B post-transfusion hepatitis (NANB PTH) was assessed. A prospective study of 192 transfusion recipients was performed to compare the incidence of NANB PTH after the introduction of the second-generation anti-HCV test with the incidence before its introduction. We used a polymerase chain reaction to detect HCV-RNA and HBV-DNA in the sera of patients with NANB PTH. The incidence of acute post-transfusion hepatitis C was 11% (8 of 71) before the screening for anti-HCV as compared with 2.5% (3 of 121) after the screening (p < 0.05). Viremia was detected within the first five weeks of infection in 10 patients with acute post-transfusion hepatitis C. However, there was no significant difference in the incidence of non-A, non-B, non-C (NANBNC) PTH before screening (3 of 71, 4.2%) compared with after screening (3 of 121, 2.5%). Usually, NANBNC PTH was not clinically important. Anti-HCV screening of blood donors significantly reduces the incidence of post-transfusion hepatitis C, but not the incidence of NANBNC PTH.

Adolescent↗

PubMed1996

Nonpeptide endothelin receptor antagonists. VII: Binding characteristics of [3H]SB 209670, a novel nonpeptide antagonist of endothelin receptors.

The data presented in this manuscript describes the binding characteristics of [3H]SB 209670, a potent nonpeptide tritium-labeled endothelin (ET) receptor antagonist. The binding of this antagonist to cloned human ETA and ETB receptors was specific, saturable and of high affinity. The apparent dissociation constants were 0.20 and 1.0 nM for ETA and ETB receptors, respectively. The maximum binding was 4.7 and 22.5 pmol/mg protein for ETA and ETB receptors, respectively. Unlike [125]ET-1, the binding of [3H]SB 209670 was reversible. The half-times (T1/2) for dissociation of this ligand from ETA and ETB receptors were approximately 60 and 10 min, respectively. Competition binding studies using [3H]SB 209670 and unlabeled agonists ET-1, ET-3 and S6c indicated that these agonists displayed similar affinities for human ETB receptors, whereas with ETA receptors, ET-1 was approximately 50-fold and 1500-fold more potent than ET-3 and S6c, respectively. Of the peptide antagonists tested, BQ123 (ETA-selective peptide antagonist), displayed Ki values of 40 and > 2300 nM for ETA and ETB, whereas RES701 (ETB-selective antagonist) displayed Ki values of > 1600 and 81 nM for ETA and ETB receptors, respectively. The nonselective peptide antagonist, PD 142893, was approximately 2-fold more potent for ETA compared with ETB receptors. Similar observations were made with nonselective nonpeptide antagonists, Bosentan, (+/-) SB 209670, SB 209670, and (-) SB 209670. All these compounds were 2 to 10 times more potent for ETA than ETB receptors.

Animals↗

PubMed1995

Interaction of streptokinase and plasminogen. Studied with truncated streptokinase peptides.

The interaction of streptokinase (SK) with human plasminogen (HPlg) was investigated using truncated SK peptides prepared by gene cloning techniques. SK(16-414) and SK(16-378) could activate HPlg as efficiently as the authentic SK. SK(60-414), which had been preincubated with SK(1-59), could also activate HPlg. SK(91-414), SK(127-414), and SK(158-414), at a concentration of one-tenth of HPlg, all failed to activate HPlg. However, the truncated SK peptides in complexes with equimolar HPlg could form amidolytically active virgin enzymes that slowly converted to human plasmin (HPlm) after a lag period of 15 min. SK(16-316) could not activate HPlg. No virgin enzyme was detected when SK(16-316) was incubated with equimolar HPlg, but the HPlg in the complex was modified to HPlm after reaction for 20 min. SK(220-414) and SK(16-251) had no ability to transform HPlg to virgin enzyme or to HPlm in equimolar complex with HPlg, although they could bind to HPlg. The functions of five regions in the SK molecule (a, Ile1-Lys59; b, Ser60-Asn90; c, Val158-Arg219; d, Tyr252-Ala316; e, Ser317-Ala378) in interaction with HPlg are deduced. Region a is important in stabilizing the conformation of the SK molecule, and region b is essential for HPlg activation. Region c is required for induction of the conformational changes of HPlg to virgin enzyme. Regions c and d are required for the conversion of HPlg to HPlm in the HPlg.SK equimolar complex. Coordination of regions c, d, and e of SK is essential for a virgin enzyme formation, and coordination of regions b, c, d and e is required for an effective SK-type HPlg activator.

Base Sequence↗

PubMed1995

Interstitial myofibroblasts in experimental renal infection and scarring.

We have examined the temporal and spatial distribution of myofibroblast-like cells, a phenotype with fibroblast and smooth muscle features, in an experimental model of renal infection. Escherichia coli organisms (10(5)) were inoculated directly into the renal cortex of Sprague-Dawley rats weighing 270 g. Saline was substituted in a control group. The animals were sacrificed at five time points up to day 24 (E. coli n = 8, controls n = 3 each interval). Myofibroblasts were identified by morphology and immunohistochemistry for alpha smooth muscle actin (alpha-SMA) and compared with staining for monocytes (ED-1), collagen III, and bromodeoxyuridine incorporation. Histological changes included a focal lesion in E. coli infected animals. Interstitial alpha-SMA staining was confined to spindle-shaped cells resembling myofibroblasts. The percent fractional area of alpha-SMA staining in the lesion increased from 0.12 +/- 0.09 at day 1 to 20.0 +/- 7.1 at day 3 (p < 0.005), decreasing progressively to 2.0 +/- 2.6 by day 24. This paralleled bromodeoxyuridine incorporation in myofibroblasts: 0.4 +/- 0.5 cells/0.25 mm2 at day 1, 105.0 +/- 36.3 at day 3, and 2.6 +/- 2.2 cells/0.25 mm2 at day 24. ED-1-positive cells increased from 374 +/- 200/0.25 mm2 at day 1 to 894 +/- 88 at day 3 (p < 0.01), declining to 230 +/- 108/0.25 mm2 by day 24. Intracellular collagen III and alpha-SMA stainings were colocalized at day 3. The fractional area of collagen III increased by day 24 (p < 0.05). In conclusion, myofibroblasts accumulate transiently during renal interstitial fibrosis and are derived at least in part from local proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1995

Thrombin-mediated down-regulation of endothelin receptors in mesangial cells coincides with the down-regulation of neutral endopeptidase activity.

Thrombin-mediated down-regulation of endothelin (ET) receptors was studied in rat glomerular mesangial cells. Overnight incubation of mesangial cells with thrombin (10 nM) resulted in a significant decrease (67%) in the number of ET receptors, with no change in affinity. Northern analysis of the mRNA from these cells showed a corresponding decrease in the ETA receptor message. Such a decrease in ET receptors could result from an increase in ET levels caused by an increase in synthesis and/or a decrease in degradation. It has been previously reported that thrombin stimulates ET production in endothelial and mesangial cells. Because ET is known to be degraded by neutral endopeptidase (NEP), which is present at high levels in the kidney, the potential effects of thrombin on NEP activity were evaluated. There was a decrease of NEP activity in mesangial cells at 16 and 24 hr after treatment with 10 nM thrombin. This effect was specific for thrombin, because NEP activity was not altered after treatment with thrombin in the presence of hirudin, an inhibitor of thrombin activity. The thrombin-mediated decrease in NEP activity correlated with a decrease in NEP protein and mRNA levels, as determined by Western and Northern analyses, respectively. To determine whether the thrombin-mediated decrease in ET receptors had a functional corollary, ET-1-stimulated intracellular calcium mobilization was measured. Overnight incubation with 10 nM thrombin resulted in a significant inhibition of ET-stimulated intracellular calcium mobilization. This effect was specific for ET, because thrombin pretreatment did not affect vasopressin-stimulated intracellular calcium mobilization in mesangial cells. These results indicate that the thrombin-mediated down-regulation of ET receptors is due, in part, to a thrombin-stimulated increase in ET resulting from the down-regulation of NEP and the reported increase in ET synthesis. In addition, pretreatment of mesangial cells with ET-1 caused a significant decrease (85%) in ET receptor number and ET-1-mediated intracellular calcium release (84%), without affecting vasopressin- or thrombin-mediated responses.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: down-regulation by 1,25-dihydroxy-vitamin D3.

Endothelins (ETs) (ET-1, ET-2, and ET-3), a family of 21-amino acid peptides, mediate a host of biological responses by binding to specific cell surface receptors termed ETA and ETB. Because a role for ET in bone remodeling has been suggested, the present study was undertaken (a) to characterize ET receptors and their responses in the rat osteosarcoma cell line ROS 17/2.8 and (b) to study their regulation by 1,25-dihydroxy-vitamin D3. Binding studies using 125I-ET-1 (a nonselective agonist) and 125I-IRL-1620 (an ETB receptor-selective agonist) indicated that these cells display high affinity ETA and ETB receptors in the ratio of 3:1. Addition of ET-1 or sarafotoxin 6c to myo-[3H]inositol-labeled cells resulted in an increase in inositol phosphate accumulation as well as in intracellular Ca2+ release, suggesting that these receptors are coupled to phospholipase C. In addition, ET-1 but not sarafotoxin 6c induced a modest increase in the expression of osteocalcin protein that was completely blocked by BQ123 (an ETA receptor-selective antagonist), indicating that activation of ETA receptors plays a role in the induction of osteocalcin. Treatment of ROS osteoblasts with 10 nM 1,25-dihydroxy-vitamin D3 for 14 hr resulted in a significant (> 50%) decrease in 125I-ET-1 and 125I-IRL-1620 binding. This decrease in binding was shown to be due to a decrease in the number of ET receptors, with no change in affinity. Although both ETA and ETB receptors were down-regulated in response to 1,25-dihydroxy-vitamin D3, only ETA receptor mRNA levels were significantly decreased, with very little change in ETB mRNA levels. These data indicate that ROS osteoblasts display both ETA and ETB receptors that are functional. Induction of osteocalcin was primarily mediated by ETA receptors, and these receptors were also down-regulated at the mRNA level by 1,25-dihydroxy-vitamin D3.

Animals↗

PubMed1994

Function of streptokinase fragments in plasminogen activation.

Several peptide fragments of streptokinase (SK) were prepared by incubating SK with immobilized human plasmin (hPlm) and purified by h.p.l.c. with a reverse-phase phenyl column. The N-terminal sequences, amino acid compositions and molecular masses of these peptide fragments were determined. The SK peptide fragment of 36 kDa consisting of Ser60-Lys387 (SK-p), was the only peptide fragment that could be tightly bound to immobilized hPlm. Another three large SK peptide fragments, SK-m, SK-n and SK-o, with molecular masses of 7 kDa, 18 kDa and 30 kDa, and consisting of Ile1-Lys59, Glu148-Lys333, Ser60-Lys333 respectively, were also obtained from the supernatant of the reaction mixture. The purified SK-p had high affinity with hPlm and could activate human plasminogen (hPlg) with a kPlg one-sixth that of the native SK. SK-o had low affinity with hPlm and could also activate hPlg, although the catalytic constant was less than 1% of the native SK. SK-n, as well as SK-m, which is the N-terminal 59 amino acid peptide of the native SK, had no activator activity. However, SK-m could enhance the activator activity of both SK-o and SK-p and increase their second-order rate constants by two- and six-fold respectively. It was concluded from these studies that (1) SK-o, the Ser60-Lys333 peptide of SK, was essential for minimal SK activator activity, (2) the C-terminal peptide of SK-p, Ala334-Lys387, was essential for high affinity with hPlm, and (3) the N-terminal 59-amino-acid peptide was important in maintaining the proper conformation of SK to have its full activator activity.

Amino Acid Sequence↗

PubMed1994

Diacylglycerol formation induced by human plasmin in cultured endothelial cells.

Treatment of cultured bovine carotid artery endothelial cells with 10(-7) M plasmin increased the cellular diacylglycerol which was determined by the formation of [3H]palmitate-labeled diacylglycerol and diacylglycerol mass. Upon the stimulation with plasmin, a gradual increase in diacylglycerol formation was observed within 20 min then slightly declined. The maximal effect during the 1-h time course study was 45 and 55% increases in [3H]palmitate-labeled diacylglycerol and diacylglycerol mass, respectively, at 20 min after plasmin treatment. Formation of phosphatidylethanol was also studied in [3H]palmitate-prelabeled cells in the presence of ethanol. Treatment with plasmin for 20 min induced a significant 45% increase in phosphatidylethanol formation. The present results indicate that the plasmin-induced diacylglycerol formation in endothelial cells was at least in part mediated through the phospholipase D activation.

Animals↗

PubMed1994

Cloning and characterization of a novel endothelin receptor from Xenopus heart.

Endothelin (ET) receptors display subtype heterogeneity and so far three subtypes of ET receptors, namely ETA, ETB, and ETC, have been identified, cloned, sequenced, and characterized. Based on the binding profile of ET and related peptides, a novel ET receptor (ETAX) was identified in the follicular membranes of Xenopus laevis oocytes (Kumar, C. S., Nuthulaganti, P., Pullen, M., and Nambi, P. (1993). Mol. Pharmacol. 44, 153-157). Here we report the cloning and characterization of this ETAX subtype from X. laevis heart. A cDNA was isolated that encodes a protein of 415 amino acids that shares 74, 60, and 51% identities with human ETA, human ETB, and Xenopus ETC receptors, respectively. Competition binding studies of the cloned receptor expressed in COS cells using ET-related peptides suggested that this receptor is pharmacologically identical to that expressed in Xenopus oocyte follicular, heart, and lung membranes. Phosphoinositide turnover and oocyte electrophysiological studies indicated that the cloned receptor is functionally coupled to a second messenger system.

Amino Acid Sequence↗

PubMed1994

Human AT1 receptor is a single copy gene: characterization in a stable cell line.

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430 +/- 15 fmol/mg] exhibiting high affinity [KD = 0.15 +/- 0.02 nM] for [125I][SAR1, Ile8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed receptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5'-(gamma-thio) triphosphate [GTP gamma S]. Angiotensin II evoked a rapid efflux of 45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

Animals↗