A dramatic enhancing effect of InBr3 towards the oxidative Sonogashira cross-coupling reaction of 2-ethynylanilines.

The addition of InBr3 to the oxidative Sonogashira cross-coupling reaction of 2-ethynylaniline with (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane led to a dramatic increase in the reactivity to afford the corresponding 1,3-enynes bearing a trifluoromethyl group on their terminal sp(2) carbon. The subsequent cyclization of these 1,3-enynes under palladium catalysis provides access to the corresponding indoles bearing a 3,3,3-trifluoroprop-1-enyl group at their 2-position.

Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae.

AIMS: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1). CONCLUSIONS: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. SIGNIFICANCE AND IMPACT OF THE STUDY: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae.

AIMS: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co(2+)-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. CONCLUSIONS: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co(2+)-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.

The myocardial Na(+)-H(+) exchange: structure, regulation, and its role in heart disease.

The Na(+)-H(+) exchange (NHE) is a major mechanism by which the heart adapts to intracellular acidosis during ischemia and recovers from the acidosis after reperfusion. There are at least 6 NHE isoforms thus far identified with the NHE1 subtype representing the major one found in the mammalian myocardium. This 110-kDa glycoprotein extrudes protons concomitantly with Na(+) influx in a 1:1 stoichiometric relationship rendering the process electroneutral, and its activity is regulated by numerous factors, including phosphorylation-dependent processes. There is convincing evidence that NHE mediates tissue injury during ischemia and reperfusion, which probably reflects the fact that under conditions of tissue stress, including ischemia, Na(+)-K(+) ATPase is inhibited, thereby limiting Na(+) extrusion, resulting in an elevation in [Na(+)](i). The latter effect, in turn, will increase [Ca(2+)](i) via Na(+)-Ca(2+) exchange. In addition, NHE1 mRNA expression is elevated in response to injury, which may further contribute to the deleterious consequence of pathological insult. Extensive studies using NHE inhibitors have consistently shown protective effects against ischemic and reperfusion injury in a large variety of experimental models and has led to clinical evaluation of NHE inhibition in patients with coronary artery disease. Emerging evidence also implicates NHE1 in other cardiac disease states, and the exchanger may be particularly critical to postinfarction remodeling responses resulting in development of hypertrophy and heart failure.

Quantification of mentolabial profile using lateral roentgen cephalography and profile reconstruction by mandibular osteotomy in healthy adults and patients with scar contracture following severe facial burns.

Here mentolabial form including lateral facial contour and bony contour in healthy adults was studied to refer to values of normal mentolabial form using lateral roentgen cephalography. In soft tissue of healthy adults, thickness of mentolabial soft tissue was greater for men than for women. Degree of mentolabial curvature was greater for men than for women. In severe facial burn patients, the micrognathia-like deformity was evaluated compared with the reference values. In soft tissue of severe facial burn patients, loss of mentolabial sulcus and eversion of the lower lip were characteristically analyzed, suspecting that the major cause was scar contracture. These statistical data contribute to reconstruction of micrognathia-like deformity after severe facial burn to lead to favorable mentolabial form. It is shown that osteotomized genio-advancement produced ideal mentolabial form in severe facial burn patients, who had not been satisfied with the form by conventional operative methods, free skin flap, skin flap or free skin graft. And that the analyzed data, especially in the parameter of thickness of mentolabial sulcus, was improved.

Methionine dependency of cell growth in normal and malignant hematopoietic cells.

Normal and malignant hematopoietic cell lines, with the exception of MOLT-3, were unable to grow in the methionine-depleted medium which was supplemented with homocysteine. Normal lymphocytes were less stimulated by mitogens in the medium containing homocysteine. Colony-forming units in culture from normal bone marrow cells were not formed in the medium in which homocysteine was substituted for methionine. These data suggest that there are no differences in the methionine requirement for cell growth between normal and malignant hematopoietic cells.

Neutrophils responded to immobilized lipopolysaccharide in the absence of lipopolysaccharide-binding protein.

Lipopolysaccharide (LPS) in solution primes neutrophils for enhanced release of superoxide in response to N-formyl-methionyl-leucyl-phenylalanine. We show that LPS immobilized on polystyrene or polypropylene acted on neutrophils by a mechanism different from that of LPS in solution. Coating the surface with 1% plasma, either before coating with LPS (plasma/LPS) or after coating with LPS (LPS/plasma), was essential to induce the LPS response in neutrophils. However, plasma could be replaced by fibrinogen, type I collagen or type IV collagen, or, to a lesser extent, by fibronectin or vitronectin, which was not true for LPS in solution. About 20% of the LPS added was immobilized on the plastic surfaces, based on its ability to adsorb anti-LPS antibody after extensive washing. The amount of soluble LPS that might have been released from surfaces during the incubation with neutrophils was too low to account for the priming by immobilized LPS. About 13-20 min was needed for neutrophils to become primed after incubation with immobilized LPS. Immobilized LPS induced up-regulation of CD11b/CD18 and latent alkaline phosphatase and also enhanced the adhesive response of neutrophils. Priming by immobilized LPS was inhibited by anti-CD14 antibody or by treatment of neutrophils with the LPS antagonist LA-14-PP. When immobilized LPS was treated with anti-LPS-binding protein (LBP) antibody, the response of neutrophils to LPS/plasma was inhibited but the response to plasma/LPS or fibrinogen/LPS was not. Thus, the LPS in plasma/LPS or fibrinogen/LPS acted on neutrophils in an LBP-independent manner. We conclude that the CD14-dependent LPS receptor system of neutrophils was capable of working in the absence of LBP, but only when LPS was immobilized on a surface coated with protein.

An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma.

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl-Met-Leu-Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA-14-PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA-14-PP or Rs-LPS inhibited LPS-induced responses. When neutrophils were exposed to LA-14-PP or Rs-LPS for 3 min and then to Escherichia coli-LPS, the antagonists inhibited priming for superoxide release, and also blocked up-regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli-LPS or plasma, and was not observed at 0 degrees C, suggesting that E. coli-LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA-14-PP or Rs-LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma-dependent and CD14-dependent manner, but were not blocked by LA-14-PP or Rs-LPS. When sub-optimal concentrations of plasma were exposed to LA-14-PP or Rs-LPS, and then mixed with Pg-LPS or Bp-LPS, followed by incubation with neutrophils, priming and up-regulation of CD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS-binding protein (LBP) in plasma to immobilized E. coli-LPS was inhibited by pre-incubation of plasma with LA-14-PP or Rs-LPS. Together with the result that treatment of plasma with anti-LBP antibody abolished the cofactor activity of plasma, these results indicated that LA-14-PP and Rs-LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA-14-PP and Rs-LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.

Keratinizing squamous epithelium associated with Syringocystadenoma papilliferum differentiates towards infrainfundibulum: case report with immunohistochemical study of cytokeratins.

We describe a case of Syringocystadenoma papilliferum (SCAP) with keratinizing squamous epithelium in a 26-year-old female presenting with a dark brown to black nodule on her forehead. After surgical excision, the specimen was examined immunohistochemically using antibodies against cytokeratin (CK) 1, 8, 10, 14, 17, 18 and 19. Within the keratinizing squamous epithelium, CK1, 10, 14 and 17 were present, whereas the other CKs were absent. Based on CK expression, keratinizing squamous epithelium in SCAP seems to differentiate towards the infrainfundibulum.

Tissue reaction at the implantation of recombinant human bone morphogenetic protein-2 into the skeletal muscle.

Bone morphogenetic protein (BMP) is a unique cytokine that induces bony tissue in soft tissue. Tissue reactions at and around the implantation of recombinant human BMP-2 (rhBMP-2) into the soft tissue of rats and nonhuman primates were investigated. At the osteoinduced site of rats, massive trabeculae-lined osteoblasts and rich marrow were observed. At and around the nonosteoinduced sites of nonhuman primates, large clear nuclei were observed in reaction to rhBMP-2 implantation. The surrounding area was visually classified into zones 1, 2 and 3. Zone 3 was near the center of the implant. The area of nuclei, the major axis, the minor axis and the ratio of minor axis per major axis were image-analyzed in the histological views. In zones 1, 2 and 3, the nuclear areas were 18.0 (3.1) mean (SD); unit micron2, 33.4 (5.61) and 110.1 (23.7), respectively. The major axes of nuclear ellipses were 7.45 (0.22) (unit micron), 7.76 (0.26), and 13.9 (1.88), respectively. The minor axes were 3.07 (0.53), 5.59 (0.95) and 10.1 (1.35), respectively. The ratios of minor axis per major axis of nuclear ellipses were 0.4 (0.57), 0.72 (0.11) and 0.73 (0.11) in zones 1, 2 and 3, respectively. These results showed that in zones 2 and 3 cell and tissue reactions were marked against rhBMP-2 implantation.

Impairment of grafts by short-term warm ischemia in rat liver transplantation.

The mechanism of warm ischemic damage was investigated by assessing hepatic energy metabolism, mitochondrial functions, and lipid peroxidation (LP) of transplanted liver grafts in rats. Donor livers were stored ischemically either for 90 min at 4 degrees C (control) or for 20 min at 37 degrees C and 70 min at 4 degrees C (warm ischemia). In the control group, adenosine 5'-triphosphate (ATP) recovered within 8 min to 86% of the normal preischemic value (10.30, SEM 0.26 mumol/g dw). Total adenine nucleotides (TAN) recovered to 14.83 (SEM 0.22) mumol/g dw within 30 min, as compared with a normal level of 15.44 (SEM 0.36) mumol/g dw. The energy charge potential (ECP) immediately recovered to 0.79 (SEM 0.01) within 8 min (normal, 0.81, SEM 0.01). Mitochondrial phosphorylation rate (PR) was not significantly altered. LP averaged 451 (SEM 10) nmol/g dw in normal livers and did not change even during reperfusion (504, SEM 79, nmol/g dw, at 15 min). In contrast, in the warm ischemic group, ATP recovered only to 65% of the normal value even at 30 min (P less than 0.01), and TAN remained significantly lower than the control value (12.39, SEM 0.47, mumol/g dw, P less than 0.001). PR was normal at the end of warm ischemia, was significantly reduced at the end of the total ischemic period (P less than 0.001 and P less than 0.01, as compared with control and normal values, respectively), and gradually recovered over 30 min. LP increased and reached the maximum of 795 (SEM 84) nmol/g dw at 15-min reperfusion (P less than 0.05). In grafts treated with 50 mg/kg bw allopurinol (i.v.) 10 min prior to the onset of warm ischemia, ATP and ECP recovered to normal values at 30 min, and TAN was significantly higher than in the warm ischemic group (13.28, SEM 0.28, mumol/g dw, P less than 0.05). PR was maintained at normal values, and LP was increased but to a lesser degree than in the ischemic group. It is concluded that the delayed recovery of ATP metabolism in the warm ischemic group might be due to the loss of adenine nucleotides and the decreased PR, and that allopurinol has a protective effect against warm ischemic damage.

Pharmacology of a non-selective ETA and ETB receptor antagonist, TAK-044 and the inhibition of myocardial infarct size in rats.

1. The aims of the present study were to characterize the pharmacological profile of a new endothelin (ET) receptor antagonist, TAK-044 and to consider whether it limits the extension of myocardial infarct size in rats. 2. Binding of [125I]-ET-1 to ET receptors on rabbit ventricular and cerebellar membrane fractions was inhibited by TAK-044 with IC50 values of 3.8 nM and 130 nM, respectively. 3. It inhibited ET-1, ET-2 and ET-3-induced vasoconstriction of porcine isolated coronary arteries in a competitive (ET-1, ET-2) and a non-competitive (ET-3) manner. 4. In the rat in vivo, the ET-1-induced blood pressure changes including transient hypotension followed by sustained hypertension, were inhibited by TAK-044 (0.1-10 mg kg-1, i.v.) in a dose-dependent manner. 5. Acute myocardial infarction induced by 1 h coronary occlusion followed by 24 h reperfusion in rats caused an infarct size of 60 +/- 2% (n = 12) of the area-at-risk by weight. 6. Intravenous injection of TAK-044 10 min before coronary occlusion reduced the infarct size in a dose-dependent manner: 32% and 54% reductions at 1 and 3 mg kg-1, respectively. 7. TAK-044 administered 10 min before or 1 h after reperfusion (1 mg kg-1, i.v.) showed similar inhibitory effects: 34% and 23% reductions, respectively. 8. We conclude that TAK-044 is an ETA/ETB receptor antagonist which shows strong inhibitory effects on the extension of myocardial infarct size after coronary artery occlusion-reperfusion in rats.

Pulmonary amyloidosis with pulmonary arteriovenous fistula.

A 77-year-old man was found to have multiple pulmonary amyloidoma and arteriovenous (AV) fistula of the lungs. Massive deposits of amyloid were present in the vascular wall, which apparently caused the fistula.

Eradication of poliomyelitis: progress in the People's Republic of China.

China and the other countries of the Western Pacific Region have a goal of eradication of wild poliovirus by the end of 1995. In this report we examine the progress made toward eradication through the end of 1993. We examined the information about poliomyelitis and wild poliovirus based on the acute flaccid paralysis surveillance system. The number of reported poliomyelitis cases decreased from 4623 cases in 1989 and 5065 cases in 1990, which occurred during a large nationwide poliomyelitis epidemic, to 538 cases in 1993. Mass supplemental immunization sessions were conducted during the 1991 to 1992 and 1992 to 1993 winters. After the two rounds of supplemental immunizations in the 1992 to 1993 winter, wild poliovirus was not detected for the subsequent 21 months in 22 contiguous provinces in central and northern China, in which 980 million persons reside. In 1993 wild poliovirus was detected in only 5 provinces in southern China and in 2 provinces in the remote Western region; these provinces have only 14% of the total population in China. China is close to achieving its 1995 poliomyelitis elimination goal. Mass supplemental immunizations in children 0 to 3 years old can rapidly eliminate wild poliovirus from large, very densely populated areas, low income rural areas and remote mountainous areas. There appears to be no technical obstacle, even in the most difficult areas, to achieving global eradication of wild poliovirus by the year 2000.

Transcript of a homolog of aflR, a regulatory gene for aflatoxin synthesis in Aspergillus parasiticus, was not detected in Aspergillus oryzae strains.

Some strains of Aspergillus oryzae were shown to have homologs of aflR, a regulatory gene for aflatoxin synthesis in Aspergillus parasiticus. Transcription of an aflR homolog was examined in six strains of A. oryzae having the homologs, using polymerase chain reaction (PCR) coupled with reverse transcription. No PCR product was obtained when the RNA prepared from the A. oryzae strains cultivated under aflatoxin-producing condition was used as template for amplification of the aflR cDNA. By contrast, a PCR product of the expected size was obtained with RNA from A. parasiticus NIAH-26 processed by the same procedure. From genomic DNA of these strains, PCR products of the same size as above were obtained. Possible degradation of the aflR mRNA in the RNA preparation of the A. oryzae strains was negligible, because the calmodulin transcript was detected by PCR from the same RNA samples. Thus, the aflR homologs in the non-aflatoxigenic A. oryzae strains examined are not expressed even under aflatoxin-producing condition.

The effect of the kappa-opioid receptor agonist CI-977 in a rat model of focal cerebral ischaemia.

The effect of a novel, highly potent and selective kappa-opioid receptor agonist CI-977 upon ischaemic brain damage and brain swelling has been examined in a rat model of focal cerebral ischaemia. Focal ischaemia was produced by the permanent occlusion of the left middle cerebral artery (MCA) during a brief period of halothane anaesthesia. The animals were sacrificed 24 h after MCA occlusion and the amount of ischaemic brain damage and swelling was assessed in coronal sections at 8 predetermined stereotactic planes. Treatment with CI-977 (0.03, 0.3 or 3 mg/kg), initiated 30 min prior to MCA occlusion (and at multiple times thereafter) produced dose-dependent reductions in the volumes of infarction and of brain swelling, with the most marked reductions being noted with CI-977 (0.3 mg/kg) in both infarction (reduced by 38% from controls; P less than 0.02) and swelling (reduced by 31%; P less than 0.002). There was an excellent correlation between the volume of brain swelling and ischaemic damage which was similar with saline-treated and CI-977-treated animals (overall correlation coefficient r = 0.896). These results indicate that CI-977 is effective in reducing infarction in a model of focal cerebral ischaemia, and that the reduction in brain swelling occurs in parallel with the reduction in ischaemic damage.

Effect of the kappa-1 opioid agonist CI-977 on ischemic brain damage and cerebral blood flow after middle cerebral artery occlusion in the rat.

The effects of the kappa-1 opioid agonist CI-977 upon the volume of ischemic brain damage (defined using quantitative neuropathology) and local cerebral blood flow (CBF) (defined using quantitative [14C]iodoantipyrine autoradiography) have been examined at 4 h and 30 min, respectively, after permanent middle cerebral artery (MCA) occlusion in halothane-anesthetised rats. Treatment with CI-977 (0.3 mg/kg, s.c.) 30 min before and 30 min after occlusion of the MCA reduced the volume of infarction in the cerebral hemisphere (reduced by 27% when compared to vehicle; P < 0.05) and cerebral cortex (reduced by 32%; P < 0.05), despite a marked and sustained hypotension, with only minimal effect on damage in the caudate nucleus. In the hemisphere contralateral to the occluded MCA, treatment with CI-977 (0.3 mg/kg, s.c.) 30 min prior to the induction of ischemia failed to demonstrate any significant effect on either the level of local CBF in any of the 25 regions examined or on the volume of low CBF determined by frequency distribution analysis. In the hemisphere ipsilateral to MCA occlusion, CI-977 failed to produce statistically significant alterations in either the level of local CBF in 23 of the 25 regions or on the volume of low CBF, but areas of hyperemia were observed in both the medial caudate nucleus and lateral thalamus (local CBF increased by 65% and 86%, respectively, when compared to vehicle).(ABSTRACT TRUNCATED AT 250 WORDS)

Focal cerebral ischemia in the cat: pretreatment with a kappa-1 opioid receptor agonist, CI-977.

The effects of the kappa-1 opioid receptor agonist (5R)-(5 alpha,7 alpha,8 beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]-4- benzofuranacetamide monohydrochloride (CI-977) upon ischemic brain damage have been examined in 15 halothane-anesthetized cats. Focal cerebral ischemia was produced by permanent occlusion of one middle cerebral artery (MCA), and the animals killed 6 h later. The amount of early ischemic brain damage was assessed in coronal sections at 16 predetermined stereotactic planes. Pretreatment with CI-977 (0.3 mg/kg i.v. followed by continuous infusion at 0.15 mg/kg/h until death), initiated 15 min prior to MCA occlusion, significantly reduced the volume of ischemic brain damage (from 2345 +/- 675 mm3 of the cerebral hemisphere in vehicle-treated cats to 1569 +/- 370 mm3 in CI-977-treated cats; P < 0.01). These data indicate that the kappa-1 opioid agonist CI-977 is neuroprotective in a model of focal cerebral ischemia in a gyrencephalic species where key systemic variables have been assessed throughout the entire post-ischemic period.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart.

The effects of the endothelin receptor antagonist TAK-044 (cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-L-ala nyl-L-alpha-aspartyl-D-2-(2-thienyl)glycyl-L-leucyl-D-tryptophyl]+ ++disodiu m salt) and BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-1-induced responses (100 pmol/heart) with IC50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1-30 mu M) partially inhibited the endothelin-1-induced hypertension (30-40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10 mu M) nor Nomega-nitro-L-arginine methyl ester (100 mu M) attenuated the endothelin-1-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [125I]endothelin-1 binding to endothelin ET(A) receptors with IC50 values of 0.39 +/- 0.6 and 36 +/- 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET(B) receptor subtype (IC50 value: 370 +/- 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET(A) and endothelin ET(B) receptors, all of which are sensitive to TAK-044.