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HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK1 cells. At concentrations from 0.1 to 100 μg/ml (= 2.5 × 10(-8) - 2.5 × 10(-5) M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10(-8) M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10(-5) M sodium nitroprusside. HS-142-1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulation of cyclic GMP in LLC-PK1 cells through interacting with guanylyl cyclase-linked receptors.

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin (FN). FN contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the natriuretic peptide receptor A (NPR-A) sequence contains both RGD and DDX sequences. The goal of the current investigation was to determine potential interactions between FN and NPR-A on BNP induction of cGMP in cultured human cardiac fibroblasts (CFs). Further, we sought to determine whether a Mayo designed NPR-A specific RGD peptide could modify this interaction. Here we reconfirm the presence of all three natriuretic peptide receptors (NPR) in CFs. CFs plated on FN demonstrated a pronounced increase in cGMP production to BNP compared to non-coated plates. This production was also enhanced by the NPR-A specific RGD peptide, which further augmented FN associated cGMP production. Addition of HS-142-1, a NPR-A/B antagonist, abrogated the responses of BNP to both FN and the NPR-A specific RGD peptide. Finally, we defined a possible role for the NPR-C through non-cGMP mechanisms in mediating the anti-proliferative actions of BNP in CFs where the NPR-C antagonist cANF 4-28 but not HS-142-1 blocked BNP-mediated inhibition of proliferation of CFs. We conclude that NPR-A interacts with components of the ECM such as FN to enhance BNP activation of cGMP and that a small NPR-A specific RGD peptide augments this action of BNP with possible therapeutic implications. Lastly, the NPR-C may also have a role in mediating anti-proliferative actions of BNP in CFs.

The dedifferentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the progression of atherosclerosis and restenosis after angioplasty. Thus, factors that stimulate smooth muscle cell differentiation should be useful for therapy for these diseases. Previously, we found that l-ascorbic acid (L-Asc) induces the expression of smooth muscle-specific genes in a pluripotent bone marrow stromal cell line, TBR-B. This finding suggests that l-Asc stimulates the differentiation of smooth muscle cells. In the present study, we investigated the effects of l-Asc and its derivatives on the differentiation state of VSMCs in vitro and in vivo. l-Asc and its long-lasting derivatives stimulated the production of smooth muscle-specific myosin heavy chain-1 (SM1) and calponin 1 in a dose-dependent manner in rat cultured VSMCs, and the elevated production of SM1 and calponin 1 was maintained for at least 2 weeks. Moreover, oral administration of 3 g/kg of l-Asc to the balloon-injured rats induced a higher expression of SM1 and calponin 1 in the injured arteries compared with that from administration of the delivery vehicle alone. These data demonstrated new biologic activity, such as the stimulation of VSMC differentiation, of l-Asc and its long-lasting derivatives. In addition, these compounds may serve as useful tools for analysis of the differentiation of VSMCs and for therapy for vascular diseases.

EI-2128-1, a novel interleukin-1beta converting enzyme (ICE) inhibitor, was isolated from the culture broths of Penicillium sp. E-2128. EI-2128-1 selectively inhibited human recombinant ICE activity with IC50 value of 0.59 microM, without inhibiting elastase and cathepsin B. EI-2128-1 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 value of 0.28 microM.

EI-1941-1 and -2 isolated from the culture broths of Farrowia sp. selectively inhibited the human recombinant ICE activity with IC50 values of 0.086 and 0.006 microM, respectively, without inhibiting elastase and cathepsin B. EI-1941-1 and -2 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 values of 5.0 and 10.3 microM, respectively. Biochemical characterizations of EI-1941-1 and -2 are described in this article.

We have established a novel smooth muscle cell line (SVS) which retains the expression of specific markers for smooth muscle cells, such as smooth muscle myosin-1, calponin and SM22 alpha, from temperature-sensitive SV40 large T-antigen transgenic mice. SVS cells showed temperature-dependent growth and the expression of smooth muscle markers, showing that the differentiation stage can be controlled in this cell line by culture temperature. We have constructed luciferase-reporter cell lines for calponin and SM22 alpha genes by using SVS cells. We detected some compounds including ascorbic acid, which stimulated the expression of smooth muscle markers by the reporter assay. Ascorbic acid and its long lasting derivatives stimulated the expression of markers continuously. Moreover, ascorbic acid activated the expression of markers in synthetic SMCs seen in the intima of a rat balloon injury model. Thus, this reporter system should be useful for discovery of new antiatherosclerotic drugs.

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).

1.A family of structurally related but genetically distinct natriuretic peptides exist which include atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of myocardial cell origin and C-type natriuretic peptide (CNP) of endothelial and renal epithelial cell origin. All three exert actions via cGMP, with ANP and BNP functioning via the natriuretic peptide A receptor and CNP via the natriuretic peptide B receptor. 2.Circulating and urinary natriuretic peptides were determined in response to acute intravascular volume overload (AVO). Additionally, their functional role in cardiorenal regulation during AVO was investigated by utilizing the natriuretic peptide receptor antagonist HS-142-1. Control (n = 5) and study dogs (HS-142-1, n = 9) underwent AVO with normal saline equal to 10% of body weight over 1 ;h. Both groups demonstrated similar significant increases in right atrial pressure, pulmonary capillary wedge pressure, pulmonary artery pressure and cardiac output. Circulating ANP paralleled increases in right atrial pressure and pulmonary capillary wedge pressure, with no changes in plasma BNP or CNP. At peak AVO, urinary CNP excretion was increased compared with baseline (7.0±4.2 versus 62±8.0 ;pg/min, P< 0.05). 3.In the HS-142-1-treated group, plasma cGMP was decreased compared with the control group (9.6±1.1 to 5.0±1.2 ;pmol/ml, P< 0.05). A significant attenuation of natriuresis (566±91 versus 1241±198 ;μEq/min, P< 0.05) and diuresis (4.8±0.7 versus 10.1±2.0 ;ml/min, P< 0.05) was also observed at peak AVO in the HS-142-1 treated group. 4.These findings support differential and selective responses of the three natriuretic peptides to AVO, in which plasma ANP and urinary CNP are markers for AVO. Secondly, these studies confirm the role of ANP and CNP but not BNP in the natriuretic and diuretic response to acute volume overload.

Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24–48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50of 20 ± 2 nm. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50of CEP-1347 for JNK1 activation was the same as the EC50for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

Treatment with a beta-adrenergic blocker (beta-blocker) in hypertension is associated with increased plasma atrial natriuretic peptide (ANP) levels despite a decrease in cardiac overload. The mechanism and pathophysiological significance of the phenomenon remain unclear. To clarify the role of the ANP system in the antihypertensive effects of the beta-blocker, we investigated the effects of carvedilol (30 mg/kg x day, orally, for 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). Plasma ANP levels showed a significant increase despite a significant decrease in blood pressure and heart rate in the carvedilol group. Although ANP messenger RNA levels in the heart did not change, messenger RNA levels of the natriuretic peptide-C (NP-C) receptor as a clearance receptor showed a significant decrease in both the aorta and lung in the carvedilol group. NP-C receptor densities were also significantly decreased in the lung in this group. The biological half-life of exogenous ANP in circulating blood was prolonged in the carvedilol group compared with that in the control group. Administration of the ANP receptor antagonist, HS-142-1, resulted in a greater increase in systolic blood pressure in the carvedilol group than in the control group. In addition, both basal and ANP-stimulated cGMP contents in the aorta were significantly higher in the carvedilol group. These results suggest that carvedilol potentiates the hypotensive action of ANP by increasing plasma ANP levels and enhancing the vascular response to ANP. These effects were closely related to the down-regulation of the NP-C receptor. The newly found mechanism seems to account for a sizable portion of the antihypertensive effects of carvedilol and could be of potential importance in the treatment of cardiovascular disease with beta-blockers.

The human alpha1,3-fucosyltransferase, Fuc-TVII, a key enzyme in the biosynthesis of selectin ligands, was expressed as a soluble protein-A chimeric form in a human B cell lymphoma cell line, Namalwa KJM-1, and purified using IgG-Sepharose. The enzymatic properties of recombinant soluble Fuc-TVII were then examined. Its enzyme activity was highest at pH 7.5, and the presence of 25 mM Mn2+ was required for full activity. Fuc-TVII exhibits an acceptor specificity restricted to alpha2,3-sialylated type 2 oligosaccharides, and the apparent Km values for alpha2,3-sialyl lacto-N-neotetraose and GDP-fucose were 3.08 mM and 16.4 microM, respectively. The inhibitory effects of various nucleotides on the activity of Fuc-TVII reflected its donor specificity for the nucleotide portion of GDP. Fuc-TVII was demonstrated to be useful for the synthesis of a sialyl Lewis x hexasaccharide from lacto-N-neotetraose in combination with an alpha2, 3-sialyltransferase, ST3Gal IV. Polyethylene glycols enhanced the thermal stability of Fuc-TVII, leading to increased formation of the reaction product.

A human alpha-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 micrograms/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG-Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed alpha-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal alpha-2,3-linked sialic acid among various acceptors. The apparent Km values of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.

N-Terminal cyclic peptide of RES-701-1, an endothelin (ET) type B receptor selective antagonist, was chemically combined to C-terminal peptide of ET family. Among a series of the hybrid peptide synthesized, RES-701-1(1–10)/[A15]ET-1(12–21) hybrid peptide possessed both a potent binding activity with an IC50 value of 0.24nM and a selectivity for type B receptor.

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

Novel compounds MS-681a, b, c and d were isolated from the culture broth of a fungal strain KY6568. The strain was identified as Myrothecium sp. from its morphological characteristics. MS-681a, b, c and d inhibited the activity of purified smooth muscle myosin light chain kinase with IC50 values of 0.11, 0.29, 0.095 and 0.26 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited at 100 microM by MS-681 compounds.

Atrial and brain natriuretic peptides exert renal and cardiovascular actions through binding to the natriuretic peptide-A receptor, while C-type natriuretic peptide mediates actions that occur through binding to the natriuretic peptide-B receptor, with subsequent generation of cyclic guanosine monophosphate. This study determined responses of circulating atrial natriuretic peptides in experimental acute heart failure and addressed the hypothesis that elevated circulating atrial natriuretic peptides serve a homeostatic role in regulating sodium excretion and that this action is localized to the glomerulus and distal nephron, sites rich in natriuretic peptide-A receptors.Studies were performed in the absence and presence of HS-142-1, an inhibitor of the natriuretic peptide receptors. Two groups of anesthetized dogs underwent induction of acute heart failure by rapid ventricular pacing, as characterized by decreases in cardiac output and increases in filling pressures with associated elevation of endogenous atrial natriuretic peptides secondary to increases in atrial stretch. In group 1 (n = 5, vehicle intrarenal bolus), despite acute heart failure-mediated decreases in cardiac output, sodium excretion was preserved with maintenance of the glomerular filtration rate and distal fractional sodium reabsorption. In group 2 (n = 5), in response to the natriuretic peptide receptor antagonist, HS-142-1 (0.5 mg/kg intrarenal bolus), sodium excretion (17.0 +/- 4.4 to 5.9 +/- 3.2 microEq/min; P.05) and glomerular filtration rate decreased (33.0 +/- 3.6 to 21.0 +/- 3.9 mL/min; P.05) and distal fractional sodium reabsorption increased (98.0 +/- 0.63 to 99.3 +/- 0.25%; P.05), in association with a decrease in plasma cyclic guanosine monophosphate (13.0 +/- 3.5 to 6.6 +/- 2.9 pmol/mL; P.05) and renal cyclic guanosine monophosphate generation (1,216 +/- 421 to 466 +/- 208 pmol/min; P.05).This study supports a functionally significant role for the endogenous natriuretic peptide system in preserving sodium homeostasis and glomerular filtration rate in acute heart failure.

C-terminal substituted analogs of an ETB receptor specific antagonist RES-701-1 were prepared and evaluated of their receptor binding activities. C-terminal Trp deletion or substitutions for other aromatic amino acids, containing D-configurational ones, proved to be possible without reducing the receptor binding activity, which differs from the case of ET-1 previously reported on its Trp21 for its biological activities.

EI-1511-3, -5 and EI-1625-2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1511 and E-1625. EI-1511-3, -5 and EI-1625-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.09, 0.38 and 0.2 microM, respectively. Taxonomy, fermentation of the producing strain and isolation of EI-1511-3, -5 and EI-1625-2 are described.

EI-1507-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1507. EI-1507-1 and EI-1507-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.23 and 0.42 microM, respectively. EI-1507-1 and EI-1507-2 also inhibited mature interleukin-1 beta secretion from THP-1 cells with IC50 values of 1.1 and 1.4 microM, respectively.

The effects of an endothelin (ET)-receptor B-specific antagonist, RES−701−1, on ET-induced contraction of guinea-pig trachea and on ET-induced bronchoconstriction in anaesthetized guinea-pigs were investigated. In the epithelium-removed tracheal preparation, 1 times 10−5 M RES−701−1 inhibited contractions induced by the ETB-specific agonist sarafotoxin S6c (pKB = 6ṁ10). In the epithelium-intact tracheal preparation, RES−701−1 (1 times 10−5 M) inhibited the ET−3−induced contraction (pKB = 5ṁ27), but enhanced the ET−1−induced contraction significantly and shifted the concentration-response curve to the left. The maximal responses of ET−1- and ET−3−induced contraction were augmented by epithelium removal by 1ṁ5 and 1ṁ8−fold, respectively. Against ET−3−induced contraction in the tracheal preparation without epithelium, RES−701−1 (0ṁ3−10 times 10−6 M) antagonized the contraction in a concentration-dependent manner (pA2 = 5ṁ9). On the other hand, RES−701−1 (1 times 10−5 M) did not affect ET−1−evoked responses in the trachea without epithelium. The intravenous administration of ET−1 (1ṁ5nmol kg−1) or ET−3 (1ṁ5 nmol kg−1) evoked a biphasic, fast and sustained bronchoconstriction in anaesthetized guinea-pigs pretreated with propranolol (1ṁ0 mg kg−1). When administered intravenously, RES−701−1 (0ṁ3 or 1ṁ0 mg kg−1) showed significant reduction in both phases of bronchoconstriction induced by ET−3. As in the case of ET−1−induced bronchoconstriction, RES−701−1 augmented the sustained phase although a significant reduction of the fast phase was observed. These results indicate that RES−701−1 can inhibit the ET−3−induced airway responses not only in-vitro but also in-vivo.

A novel cyclic peptide, MS-271, was isolated from the culture broth of an actinomycete, Streptomyces sp. M-271 as an inhibitor of smooth muscle myosin light chain kinase (MLCK). MS-271 inhibited the MLCK from chicken gizzard with an IC50 value of 8 microM. MS-271 did not inhibit cyclic AMP-dependent protein kinase, protein kinase C or calcium/calmodulin-dependent cyclic nucleotide phosphodiesterase at concentrations up to 400 microM. The primary structure of MS-271 was identical to that of siamycin I, an anti-HIV peptide isolated from a microbial source.

The three-dimensional structure of the endothelin B receptor (ETB) selective antagonist RES-701-1 has been determined by 1H NMR in deuterated dimethyl sulphoxide. RES-701-1 consists of 16 amino acid residues with a novel internal linkage between the beta-carboxyl group of Asp9 and the alpha-amino group of Gly1. The structural calculations were carried out with the combined use of distance geometry and simulated annealing. The result indicates that RES-701-1 adopts an extraordinary folding; the 'tail' (Trp10-Trp16) passes through the 'ring' region (Gly1-Asp9). Several critical NOEs directly support this extraordinary folding. The folding of RES-701-1 turned out to be the same as that Frèchet et al. calculated for RP 71955 which possesses the same internal linkage as RES-701-1. The obtained structure suggested that the region consisting of Thr6, Ala7, Tyr14 and Tyr15 and/or, the region consisting of Asn2, Tyr14 and Tyr15 are involved in a binding with ETB.

RES-701-1 is an ET B receptor selective antagonist obtained from Streptmyces sp. (IC 50 = 10 nM). The chemically synthesized RES-701-1 which exhibits only micromolar affinity for ET B receptor was examined by 1 H NMR and FAB-MS. The results indicated that the primary structures of the synthetic and the authentic RES-701-1 are identical but the 3D structure is completely different between them.

Effects of endothelins on cytosolic Ca 2+ level ([Ca 2+ ]i) and contraction were examined in the swine pulmonary artery and vein. In the artery, endothelin-1 and endothelin-3, but not sarafotoxin S6c and IRL 1620 (300 nM each), induced transient increase followed by sustained increase in [Ca 2+ ]i and sustained contraction. These effects were inhibited by the ET A receptor antagonist, BQ-123. In the vein, endothelin-1 and endothelin-3 (300 nM each) induced sustained increase in [Ca 2+ ]i and sustained contraction whereas sarafotoxin S6c and IRL 1620 (300 nM each) transiently increased both [Ca 2+ ]i and contractile tension. The ET B receptor in the vein was desensitized by pretreatment with sarafotoxin S6c, abolishing the effects of sarafotoxin S6c and IRL 1620 without changing the effects of endothelin-1 and endothelin-3. In contrast, an ET B antagonist, RES-701-1, antagonized the effects of IRL 1620 without changing the effects of other stimulants. In both artery and vein, the maximum contraction induced by these stimulants was greater than that induced by KCl at a given [Ca 2+ ]i. In the absence of external Ca 2+ , endothelin-1 and endothelin-3 induced transient increase in [Ca 2+ ]i and slow susained contraction in both artery and vein. In the vein, sarafotoxin S6c induced small sustained contraction without changing [Ca 2+ ]i. In the permeabilized artery and vein, endothelin-1 augmented the contraction induced by Ca 2+ . These results suggest that the ET A receptors in the artery and vein are coupled to Ca 2+ release (which does not seem to trigger contraction), Ca 2+ influx and Ca 2+ sensitization. In contrast, the ET B receptor in the vein is coupled to Ca 2+ influx and Ca 2+ sensitization but not to Ca 2+ release.

We examined the potential role of myosin and actin in the release of human immunodeficiency virus type 1 (HIV-1) from infected cells. Wortmannin (100 nM to 5 microM), an effective inhibitor of myosin light chain kinase, blocked the release of HIV-1 from infected T-lymphoblastoid and monocytoid cells in a concentration-dependent manner. Cytochalasin D, a reagent that disrupts the equilibrium between monomeric and polymeric actin, also partially inhibited the release of HIV-1 from the infected cells. At the budding stage, myosin and HIV-1 protein were detected in the same areas on the plasma membrane by using dual-label immunofluorescence microscopy and immunoelectron microscopy. In the presence of 5 microM wortmannin, viral components were observed on the plasma membrane by using immunofluorescence microscopy and electron microscopy, implying that wortmannin did not disturb the transport of viral proteins to the plasma membrane but rather inhibited budding.

A novel compound MS-444 was isolated from the culture broth of a bacterial strain KY7123. The strain was identified as Micromonospora sp. from its morphological and cultural characteristics. The compound inhibited the activity of purified smooth muscle myosin light chain kinase with an IC50 value of 10 microM. The production, isolation, physico-chemical properties and biological activities of MS-444 were described in this paper.

The effects of a novel N- methyl- d -aspartate (NMDA) receptor antagonists, ES-242-1 3,3′-dimethyl-3,4,3′,4′-tetrahydro-6,8,6′8′-tetramethoxy-[10,10′-bi-2-oxanthracene]-4,9,9′(1 H ,1′ H )-triol 4-acetate, on NMDA-induced increases of intracellular Ca 2+ concentration in cultured hippocampal neurons were examined.ES-242-1 selectively blocked the NMDA-induced increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ), but not the [Ca 2+ ] i increase stimulated by quisqualate or kainate. The effect of ES-242-1 appeared in the slow development of a blockade of [Ca 2+ ] i (half blocking time: 90sec) when 100 μM NMDA was applied with 10 μM ES-242-1, whereas the initial [Ca 2+ ] i rise was attenuated by 10 μM ES-242-1 when the latter was applied with a lower concentration of NMDA (10 μM). This is consistent with a previous observation that ES-242-1 binds to both the transmitter recognition site and the channel domain. The blockade by ES-242-1 was reversed by washing. In constrast, the blockade by MK-801 was not relieved easily by washing. These results suggest that ES-242-1 blocks the NMDA-induced [Ca 2+ ] i increase due to a combination of two well-recognized mechanisms, which are different from that of MK-801, at the NMDA receptor.

RES-701-1 (cyclic (Gly1-Asp9)(Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe- Phe-Asn-Tyr-Tyr-Trp)), a peptide isolated from Streptomyces sp., has been reported to inhibit the endothelin ETB receptor. We examined the effects of this peptide on the blood vessels and the small intestine. In isolated rat aorta without endothelium, 10 microM RES-701-1 did not affect the resting tone, nor did it attenuate the contractions induced by endothelin-1, endothelin-3 or norepinephrine. In the aorta with endothelium, 3 microM RES-701-1 shifted the concentration-response curves for the contractile effects of endothelin-1 and endothelin-3 to the left. Removal of endothelium showed a similar effect to 3 microM RES-701-1. In the norepinephrine-stimulated aorta, endothelium-dependent relaxation induced by endothelin-3 was antagonized by 0.3-10 microM RES-701-1 in a concentration-dependent manner. In the guinea pig ileum stimulated by carbachol, endothelin-3 induced a transient relaxation followed by sustained relaxation. RES-101-1 (3 microM) selectively inhibited the transient relaxation. Since it has been shown that the contractile effects of endothelins in the aorta are mediated by the endothelin ETA receptor whereas the endothelium-dependent relaxation and the ileal relaxation are mediated by the endothelin ETB receptor, it is suggested that RES-701-1 is a selective antagonist against the endothelin ETB receptor.

In rabbit saphenous vein, endothelin (ET)-1 and ET-3 induced sustained contractions whereas the selective agonists of the ETB receptor, sarafotoxin S6c (STXc) and IRL 1620, induced transient contractions. In the presence of an ETA antagonist, BQ-123, contractions induced by ET-1, STXc and IRL 1620 did not change whereas ET-3 induced only transient contraction. The ETB antagonists, RES-701-1 and IRL 1038, only weakly antagonized the effects of these stimulants. In the muscle pretreated with STXc, neither STXc nor IRL 1620 was effective whereas ET-3 induced sustained contraction at higher concentrations than ET-1. In the muscle pretreated with STXc, BQ-123 weakly antagonized the effect of ET-1 and abolished the effect of ET-3. These results suggest that there are two types of ET receptors; less tachyphylactic and isopeptide-selective ETA receptor, and tachyphylactic and isopeptide-nonselective ETB receptor. The ETA receptors may be further classified as a BQ-123-sensitive ETA1 and a BQ-123-insensitive ETA2 subtypes. The ETB receptors may also be subclassified as the ETB1 and ETB2 subtypes based on the sensitivity to the ETB antagonists.

RES-701-1, a novel cyclic peptide endothelin antagonist, was isolated from the culture broth of Streptomyces sp. RE-701. RES-701-1 selectively inhibited the ET-1 binding to type B endothelin receptor (ETB receptor) with an IC50 of 10 nM expressed in CHO cells and blocked the ET-1-induced elevation of intracellular free Ca2+ concentration in ETB receptor-expressing COS-7 cells. Characterization of producing strain, fermentation, isolation, structure, physico-chemical and biological properties of RES-701-1 are described.

A novel compound, PS-990, which induces differentiation of neuroblastoma cells, was isolated from the culture broth of a fungus, Acremonium sp. KY12702. PS-990 inhibited brain calcium calmodulin-dependent cyclic nucleotide phosphodiesterase with an IC50 value of 3 micrograms/ml, and markedly induced neurite extension of mouse neuroblastoma, Neuro2A, at concentrations ranging from 10 to 30 micrograms ml.

The structure of RES-701-1, a novel microbial endothelin type B receptor antagonist, was determined. Protein chemical data and FAB-MS have identified RES-701-1 to be a cyclic polypeptide consisting of 16 common L-amino acids. This compound is so stable against proteolysis that its enzymatic digestion under standard conditions proved to be very difficult. Therefore, the primary sequence of RES-701-1 was determined by the application of advanced protein chemical methods to be Gly1-Asn2-Trp3-His4-Gly5-Thr6-Ala7-Pro8-++ +Asp9-Trp10-Phe11-Phe12-Asn13- Tyr14-Tyr15-Trp16. The compound is cyclized between the beta-carboxyl group of Asp9 and the alpha-amino group of Gly1.

MS-282a and MS-282b were isolated from the culture broth of Streptomyces tauricus ATCC 27470 as inhibitors of smooth muscle myosin light chain kinase (MLCK). MS-282a and MS-282b inhibited the activity of chicken gizzard MLCK with IC50 values of 3.8 microM and 5.2 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited by 150 microM MS-282a at all. It is likely that MS-282a blocks MLCK activity by antagonizing calmodulin since 1) the compound inhibited calmodulin-dependent but not calmodulin-independent activity of MLCK; 2) the inhibition of MLCK was antagonized by increasing concentrations of calmodulin, and 3) the compound inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase.

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.

In this report, we describe the effects of a recently described atrial natriuretic peptide (ANP) antagonist, HS-142-1, on the action of ANP on Percoll-purified mouse Leydig cells. Incubation of the Leydig cells with 10(-8) M ANP for 3 h resulted in a 16-fold stimulation of testosterone production over basal. Addition of HS-142-1 in a concentration range of 0.1 to 5 micrograms/ml resulted in a dose-dependent inhibition of ANP-induced testosterone production, a nearly complete inhibition being achieved with 5 micrograms/ml antagonist. Testosterone production by unstimulated cells or in cells stimulated with hCG was not affected by the antagonist. HS-142-1 was also able to inhibit the ANP-stimulated cyclic guanosine monophosphate (GMP) formation in the cells, in a dose-dependent manner. However, cyclic AMP level in cells stimulated with either forskolin or hCG remained unaffected by HS-142-1 even when added at a concentration of 5 micrograms/ml. Results obtained from 125I-ANP binding experiments showed that HS-142-1 is able to competitively inhibit the binding of the radioligand to its receptors on the Leydig cells. Thus evidence obtained in this study permit us to conclude that HS-142-1 is a potent and specific antagonist of ANP, has no toxic effect on the cells and is able to inhibit competitively the binding of ANP to its guanylate cyclase coupled receptors. Availability of such antagonists are likely to facilitate research on the physiology of ANP.

ES-242-1, a novel bioxanthracene, is the first microbial compound found to act with a novel mechanism of action on the N-methyl-D-aspartate (NMDA) receptor and may possess neuroprotective properties useful in the treatment of disease involving glutamate toxicity. HS-142-1 is the first compound to be discovered as a specific antagonist for functional receptors for natriuretic peptide; the compound would be useful to clarify the physiological and pathophysiological functions of natriuretic peptides. The protein kinase inhibitor K-252a was recently reported to potentiate the activity of a neurotrophin and to promote some neuronal survival. K-252a and its derivatives would be lead compounds for the development of compounds therapeutically useful in the treatment of neurological disease and injury.

MS-347a was isolated from the culture broths of Aspergillus sp. KY52178 as an inhibitor of smooth muscle myosin light chain kinase (MLCK). MS-347a inhibited the activity of chicken gizzard MLCK with an IC50 value of 9.2 microM. The inhibition was dependent on time of preincubation of MS-347a with the enzyme, suggesting irreversible inhibition. It is likely that the inhibitor binds to the catalytic domain of MLCK, since the compound inhibited not only calmodulin-dependent but also calmodulin-independent activity of MLCK. Calmodulin-dependent cyclic nucleotide phosphodiesterase, cAMP-dependent protein kinase and cGMP-dependent protein kinase were not inhibited by 150 microM MS-347a at all, although the compound inhibited protein kinase C with an IC50 value of 16 microM. MS-347b, a minor component was also isolated from the same culture broths. This minor component at 150 microM did not inhibit the activity of MLCK.

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.

A novel compound, PS-990, which induces differentiation of neuroblastoma cells, was isolated from the culture broth of a fungus, Acremonuim sp. KY12702. PS-990 inhibited brain calcium/calmodulin-dependent cyclic nucleotide phosphodiesterase with an IC50 value of 3μg/ml, and markedly induced neurite extension of mouse neuroblastoma, Neuro2A, at concentrations ranging from 10 to 30μg/ml.

RES-1214-1 and -2, novel and non-peptidic endothelin antagonists, were isolated from the cultured broth of a fungus, Pestalotiopsis sp. RE-1214. RES-1214-1 and -2 selectivity inhibited the ET-1 binding to endothelin type A receptor (ETA receptor) with IC50 values of 1.5 microM and 10 microM, respectively. RES-1214-1 and -2 inhibited the increase in intracellular Ca2+ concentration elicited by 1 nM ET-1 in A10 cells. Taxonomy of producing strains, fermentation, isolation, structural determination, and biochemical properties of RES-1214-1 and-2 are described.

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KY11784. RP-1776 selectively inhibited the binding of PDGF BB to the extracellular domain of the PDGF beta-receptor with an IC50 value of 11 +/- 6 microM. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGF beta-receptor induced by PDGF BB. These results suggested that RP-1776 antagonizes the signaling of PDGF BB probably through the inhibition of PDGF BB binding to the PDGF beta-receptor.