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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.

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2).Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment.We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.

Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.

To elucidate the mechanism of induction of apoptosis by geranylgeraniol (GGO), which is a potent inducer of apoptosis in various lines of human cancer cells, we examined the role of intracellular acidification during GGO-induced apoptosis using human leukemia HL60 cells. Flow cytometry analysis revealed that apoptosis induced in human leukemia HL60 cells by GGO was associated with intracellular acidification. Both GGO-induced intracellular acidification and apoptosis as analyzed by DNA fragmentation were inhibited by phorbol myristate acetate (TPA) and O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, but not by ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). These results suggest that the early concentration change of intracellular Ca(2+) and the corresponding decrease in intracellular pH are required for the induction of apoptosis in HL60 cells by GGO.

Medullary neurons containing pituitary adenylate cyclase-activating polypeptide (PACAP) and noradrenalin (NA) project to the hypothalamus and they are involved in the regulation of arginine vasopressin (AVP) neurons. At the ultrastructural level, PACAP immunoreactivity was detected in the granular vesicles in catecholaminergic nerve terminals that made synaptic contact with AVP neurons. Both PACAP (at least 1 nM) and NA (at least 1 microM) induced large increases in the cytosolic Ca2+ concentration ([Ca2+]i) in isolated AVP cells. PACAP at 0.1 nM and NA at 0.1 microM had little effects, if any, on [Ca2+]i. However, when 0.1 nM PACAP and 0.1 microM NA were combined, they evoked large increase in [Ca2+]i in AVP neurons. An inhibitor of protein kinase A (PKA) completely inhibited the PACAP-induced increase in [Ca2+]i, but only partly inhibited the NA-induced increase in [Ca2+]i. In AVP cells that were prelabeled with quinacrine, PACAP and NA acted synergistically to induce a loss of quinacrine fluorescence, indicating secretion of neurosecretory granules in AVP neurons. The results suggest that PACAP and NA, coreleased from the same nerve terminals, act in synergy to evoke calcium signaling and secretion in AVP neurons, and that the synergism is mediated by the interaction between cAMP-PKA pathway an as yet unidentified factor "X" linked to L-type Ca2+ channels. The synergism between PACAP and NA may contribute to the regulation of AVP secretion under physiological conditions.

We have found previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases the number of astrocytes generated from cultured mouse neural stem cells (NSCs) via a mechanism that is independent of the cyclic AMP/protein kinase A pathway (Ohno et al., 2005). In the present study, the signaling pathway involved in the differentiation process was further investigated. PACAP-induced differentiation was inhibited by the phospholipase C inhibitor, U73122, the protein kinase C (PKC) inhibitor, chelerythrine, and the intracellular calcium chelator, BAPTA-AM, and was mimicked by phorbol 12-myristate 13-acetate (PMA), but not by 4alpha-PMA. These results suggest that the PACAP-generated signal was mediated via the PACAP receptor, PAC1 stimulated heterotrimeric G-protein, resulting in activation of phospholipase C, followed by calcium- and phospholipid-dependent protein kinase C (cPKC). To elucidate the involvement of the different isoforms of cPKC, their gene and protein expression were examined. Embryonic NSCs expressed alpha and betaII PKC, but lacked PKCgamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the betaII isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKCalpha increased linearly up to Day 6. Overexpression of PKCbetaII with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominant-negative mutant of PKCbetaII proved inhibitory. These results indicate that the beta isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs into astrocytes.

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT–PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP 6-38 , had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.

beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.

Purpose. In this study, we examined the effects of vitamin K2 (menaquinone 4), which has a geranylgeranyl side chain, on various lines of cells derived from human solid tumors and compared them with the effects of geranylgeraniol (GGO). Methods. Cell proliferation was determined with 3′-[1-[(phenylamino)carbonyl]-3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the induction of apoptosis was analyzed by TUNEL staining and flow cytometry as well as by measurement of DNA fragmentation, released nucleosomes and caspase-3 activity. Levels of Bcl-2, Bax and cytochrome c were determined by immunoblotting. Results. GGO inhibited the growth of all eight cell lines derived from solid tumors, while vitamin K2 selectively inhibited the proliferation of ovarian TYK-nu and pancreatic MIA PaCa-2 cancer cells, inducing apoptosis in both cell lines. Far more time was required for the induction of apoptosis in these two cell lines by vitamin K2 than by GGO. Apoptotic signals induced in TYK-nu cells during the first 2 days that followed the addition of vitamin K2 to the culture medium were reversible, but these signals became irreversible after 3 days of treatment with vitamin K2. The induction of apoptosis in TYK-nu cells by vitamin K2 was inhibited by cycloheximide and also by starvation at a low concentration of serum. Neither cycloheximide nor starvation had any effect on the induction of apoptosis by GGO. Cytochrome c was released simultaneously with the initiation of apoptosis on treatment of TYK-nu cells with vitamin K2 or GGO. However, GGO induced the release of cytochrome c from isolated mitochondria, while vitamin K2 did not. The amount of Bcl-2 in TYK-nu cells was reduced by vitamin K2, but not by GGO. Conclusions. In contrast to GGO, vitamin K2 induced apoptosis selectively in pancreatic MIA-PaCa 2 and ovarian TYK-nu cancer cells. It is suggested that de novo protein synthesis might be necessary for induction of apoptosis by vitamin K2 but not by GGO, and thus, that vitamin K2 and GGO might induce apoptosis by different mechanisms.

Treatment of human epidermoid carcinoma A431 cells with transplatin ( trans -DDP) and β-hydroxyisovalerylshikon (β-HIVS) together had a strong inhibitory effect on A431 cells. By contrast, trans -DDP and β-HIVS by themselves, at the same respective concentrations, had practically no effect. The tyrosine kinase activities of v-Src and epidermal growth factor (EGFR) were inhibited by each of the two agents alone and strongly inhibited by combination. The observed synergistic inhibitory effect on the growth of A431 cells might have resulted from the inhibition of EGFR by trans -DDP, as well as by β-HIVS.

We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1-6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis.

Screening of various natural products in a search for novel inducers of apoptosis in human leukemia cells led us to identify the strong apoptosis-inducing activity in a fraction extracted with methanol from the roots of Sophora subprostrata Chun et T. Chen. We purified the compound that induced apoptosis in human leukemia cells and identified it as sophoranone. Sophoranone inhibited cell growth and induced apoptosis in various lines of cells from human solid tumors, with 50% inhibition of growth of human stomach cancer MKN7 cells at 1.2 +/- 0.3 microM. The growth-inhibitory and apoptosis-inducing activities of sophoranone for leukemia U937 cells were very much stronger than those of other flavonoids, such as daidzein, genistein and quercetin. At the early stages of treatment of U937 cells with sophoranone, reactive oxygen species were formed, mitochondrial permeability pores were opened and cytochrome c was released from mitochondria. Cytochrome c was also released upon treatment of isolated mitochondria with sophoranone. Inhibitors of complexes III and IV, but not complexes I and II, of the mitochondrial respiratory chain prevented the release of cytochrome c from isolated mitochondria by sophoranone, as well as the induction of apoptosis in U937 cells in response to sophoranone. Our results indicate that sophoranone might be a unique apoptosis-inducing anticancer agent that targets mitochondria.

The cytokine interleukin-1 (IL-1) has been implicated in ischemic brain damage, because the IL-1 receptor antagonist markedly inhibits experimentally induced neuronal loss. However, to date, no studies have demonstrated the involvement of endogenous IL-1alpha and IL- 1beta in neurodegeneration. We report here, for the first time, that mice lacking IL-1alpha/beta (double knockout) exhibit markedly reduced neuronal loss and apoptotic cell death when exposed to transient cardiac arrest. Furthermore, we show that, despite the reduced neuronal loss, phosphorylation of JNK/SAPK (c-Jun NH2- terminal protein kinase/stress activated protein kinase) and p38 enzymes remain elevated in IL-1 knockout mice. In contrast, the inducible nitric oxide (iNOS) immunoreactivity after global ischemia was reduced in IL-1 knockout mice as compared with wild-type mice. The levels of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in the hippocampus of wild-type mice were increased with time after ischemia-reperfusion, whereas the increase was significantly inhibited in IL-1 knockout mice. These observations strongly suggest that endogenous IL-1 contributes to ischemic brain damage, and this influence may act through the release of nitric oxide by iNOS.

Immunohistochemistry and Western blotting were used to determine the distribution of orexin receptors in the rat brain. Strong orexin receptor 1 (OX1R) immunoreactivity was detected in the hypothalamus including the arcuate, ventromedial, and tuberomammillary nuclei that are involved in feeding regulation. The neuropeptide Y- and proopiomelanocortin-containing neurons of the arcuate nucleus, which act to stimulate or to inhibit feeding, respectively, displayed intense OX1R immunoreactivity by double immunostaining. Western blotting analysis yielded a 50-kDa major band of OX1R.

Beta-hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from Lithospermium radix, most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of beta-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk-1 was weakly inhibited by beta-HIVS. The inhibition by beta-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC50 values of beta-HIVS for EGFR and v-Src were approximately 0.7 microM and 1 microM, respectively. Moreover, the inhibition of v-Src by beta-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of beta-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.

Methylmercury (MeHg) is an environmental pollutant known to cause neurobehavioral defects, and it is especially toxic to the developing brain. In contrast to the adult, the developing brain consists of a large number of dividing neural progenitor cells (NPCs), which are vulnerable targets for MeHg toxicity. In a previous study, we showed that the embryonic NPCs from the telencephalon are more sensitive to MeHg than other neural cells. Here, we investigated the mechanism of cell death underlying MeHg toxicity. We observed that exposure of NPCs to MeHg caused DNA laddering in a dose- and time-dependent manner. Decreased pro-caspase3 and increased cleaved-caspase3 protein was observed 3-12 hours after incubation of NPCs with MeHg. Moreover, the caspase-inhibitor Z-VAD FMK significantly suppressed MeHg-induced cell death in a dose-dependent manner. These results suggest that environmentally relevant levels of MeHg exposure induce apoptosis in NPCs.

Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.

We have shown previously that extracellular ATP and bradykinin can stimulate the release of mucin from cultured human pulmonary mucoepidermoid carcinoma cells (NCI-H292) (Kusano et al 1997). Using this same cell culture system, we studied the mechanism by which this ATP- and bradykinin-induced mucin release occurs. We found that the mucin secretory response to ATP was not significantly affected by extracellular Ca2+ depletion, but the response to bradykinin was significantly inhibited by Ca2+-free medium. ATP and bradykinin increased the concentration of free intracellular Ca2+ ([Ca2+]i); the ATP-stimulated response was not affected by extracellular Ca2+ depletion, the bradykinin-stimulated response was prevented in the absence of extracellular Ca2+. ATP and bradykinin enhanced the accumulation of inositol 1,4,5-triphosphate in NCI-H292 cells and pretreatment with pertussis toxin blocked the increase in mucin secretion induced by extracellular ATP or bradykinin. These results suggest that extracellular ATP and bradykinin stimulate mucin release from NCI-H292 cells by a signal transduction that seems to involve ATP- or bradykinin-activated receptors associated with phospholipase C via pertussis toxin-sensitive GTP-binding proteins. These findings may suggest a new direction of research into the regulation of airway mucin secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide and its specific receptor (the PAC(1) receptor) is widely distributed in the rat brain. It has been reported that alternative splicing of the region encoding the third intracellular loop of the PAC(1) receptor generates six isoforms which are differentially coupled to signal transduction pathways, but the precise distribution and localization of these splice isoforms in the brain remain to be determined. Using the initial specific primer pairs which correspond to the 'hip' or 'hop' types of receptors for the solution-phase reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated that the major splice variants of the PAC(1) receptor in various regions of the rat brain are the short splice isoform 'PAC(1)-R-s' which does not contain either the 'hip' or 'hop' cassette and the another splice isoform, 'PAC(1)-R-hop', which contains the 'hop' cassette. With an innovative molecular histochemical technique, in situ RT-PCR, we determined that these two splice isoforms are both intensely expressed in the mitral cells of the olfactory bulb, the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and neocortex, and many neurons in the nuclei of hypothalamus and thalamus as well as other regions. The initial mapping of the cell type-specific expression of these two splice variants of the PAC(1) receptor provides the basis for a better understanding of the functional significance of the PAC(1)-R and its ligand PACAP in various brain regions.

Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermium radix, inhibited the growth of various lines of cancer cells derived from human solid tumors at low concentrations between 10(-8) and 10(-6) M. When HL-60 cells were treated with 10(-6) M beta-HIVS for 3 h, characteristic features of apoptosis, such as DNA fragmentation, nuclear fragmentation, and activation of caspase-3-like activity, were observed. The most characteristic features of the effect of beta-HIVS were the remarkable morphological changes induced upon treatment of HL-60 cells with beta-HIVS, as visualized on the staining of actin filaments with phalloidin labeled with tetramethylrhodamine B isothiocyanate. Moreover, activation of MAP kinases, such as ERK2, JNK and p38, was detected after treatment with 10(-6) M beta-HIVS preceding the appearance of the characteristics of apoptosis, and the features of the activation of these MAP kinases were quite different from those of Fas and anticancer drug-induced apoptosis. The activation of JNK by beta-HIVS was not inhibited by inhibitors of caspases, suggesting that JNK is located either upstream or independent of the caspase signaling pathway. beta-HIVS did not inhibit the activity of topoisomerase II. These results indicate that beta-HIVS induces apoptosis in HL-60 cells through a mechanism unlike those reported for anti-Fas antibodies and etoposide.

Phosphoneuroprotein (PNP 14) is abundant in the central nervous system and is localized at nerve endings but not in synaptic vesicles. In this study, we examined the presence of PNP 14 in various endocrine tissues of the rat. PNP 14 was not detected in the endocrine cells of the intestine, testes, or adrenal gland, but it was present in axon terminals in both the medulla of the adrenal gland and the anterior pituitary gland. When testes were stained with PNP 14-specific antibodies by an indirect immunofluorescence method, PNP 14 was found in Sertoli cells of the testes, associated with fibrillar structures. PNP 14 was also detected in cultured Sertoli cells with a fibrillar pattern in the cytoplasm and around the nuclei. The fibrillar structure did not resemble actin stress fibers, microtubules, or intermediate filaments. The amount of PNP 14 in the testis changed with development. It increased markedly during the first 4 weeks after birth and then decreased. During the first 4 weeks after birth, spermatogonia undergo two rounds of meiosis. It is possible, therefore, that PNP 14 might be a factor related to meiosis.

The present study demonstrates that projections ascending from the caudal ventrolateral medulla have direct effects on the expression of the immediate early gene c-fos and of the arginine-vasopressin gene in neurosecretory cells of the hypothalamic supraoptic nucleus. Intense Fos-like immunoreactivity (Fos-LI) was observed in many magnocellular neurons of the supraoptic nucleus after electrical stimulation of the caudal ventrolateral medulla. In sham-operated rats, Fos-LI was absent or present in very few magnocellular neurons in the supraoptic nucleus. Fos-LI was visible in neurons expressing arginine-vasopressin, and was seen rarely in oxytocin neurons by double-immunostaining method. This study showed that 76% of all Fos-positive cells were arginine-vasopressin immunoreactive, whereas only 4% of them showed oxytocin immunoreactivity in the supraoptic nucleus. With in situ hybridization, a high level of arginine-vasopressin mRNA was noted in the supraoptic nucleus 3 h after stimulation of the caudal ventrolateral medulla; the expression was highest 6 h after the stimulation compared with the same region in sham-operated animals. These findings suggest that noradrenaline, released from the axon terminals originating from the caudal ventrolateral medulla, may participate in the regulation of gene transcription of arginine-vasopressin in response to physiological stimuli.

The distribution of leptin receptor in the rat brain was determined by immunocytochemistry and Western blotting. Strong leptin receptor immunoreactivity was detected in the arcuate, paraventricular and ventromedial nuclei of the hypothalamus, and lateral hypothalamic area. The olfactory bulb, neocortex, cerebellar cortex, dorsal raphe nucleus, inferior olive nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve also showed intense immunoreactivity. Western blotting analysis yielded a 120-kDa major band.

Although nicotinic acetylcholine receptors (nAChRs) have been reported to function in the cerebellar cortex, nicotinic acetylcholine receptor subtypes contributing to nicotinic currents in the cortex have not been well characterized at the subcellular level. This study deals with immunocytochemical localization of the nicotinic acetylcholine receptor alpha4 subunit using a monoclonal antibody against the alpha4 subunit. Alpha4-LI (alpha4-like immunoreactivity) was detected in the cell bodies of molecular, Purkinje cell and granular layers. In particular, the cell bodies of Purkinje cells were extensively immunostained. In Purkinje cells, alpha4-LI was found in perikarya mainly associated with rough endoplasmic reticulum, plasma membrane, and cytoplasmic matrix. At higher magnification, the immunoreaction product was densely localized along with somatic plasma membranes at the axo-somatic synapse and the plasma membranes at extrasynaptic regions of cell bodies. Alpha4-LI was also found in the axon terminals which form synapses with Purkinje cells. In the granular layer, somatic cell membranes of granular cells were immunostained. These morphological observations suggest that alpha4-containing nAChRs contribute nicotinic currents reported in Purkinje cells, and that presynaptic alpha4-containing nAChRs regulate the release of neurotransmitters on the axon terminals found near Purkinje cells.

The subcellular localization and functional significance of neuronal nicotinic acetylcholine receptor alpha4-subunits were investigated in the rat hypothalamic supraoptic nucleus. A high level of alpha4 mRNA expression was found in the magnocellular neurons in the supraoptic nucleus. Strong immunoreactitivy for alpha4 in neurons of the supraoptic nucleus was detected in the rough endoplasmic reticulum and cytoplasmic matrix, although it was very weak in the Golgi apparatus, except for the transport vesicles. Immunoreactivity for alpha4 was detected in both the pre-synaptic axon terminals and post-synaptic axon terminals. A high level of signals for vasopressin mRNA was detected in the supraoptic nucleus after the animals were injected s.c. with nicotine. These findings suggest that alpha4-containing subtypes are synthesized in the rough endoplasmic reticulum and transported to the plasma membrane and serve as pre- and post-synaptic nicotinic acetylcholine receptors. Nicotine may up-regulate vasopressin gene expression in the supraoptic nucleus, acting through nicotinic acetylcholine receptors.

When human leukemia HL-60 cells were treated with 10(-7) M bufalin, the amounts of both topoisomerase (topo) II alpha and II beta and the activity of topo II decreased markedly and were almost undetectable 18 h after the start of treatment. The level of topo II mRNA started to decrease immediately after the start of treatment with bufalin, with a subsequent decrease in the amount of topo II alpha protein. These changes preceded the fragmentation of DNA, a typical feature of apoptosis. The results suggest that bufalin caused a marked decrease in the steady-state level of topo II alpha mRNA, which led to a decrease in the amount and activity of the enzyme and to the induction of apoptosis. A reduction in the level of topo II alpha by bufalin was also observed in other lines of human leukemia cells such as ML1 and U937. The results were exploited to potentiate the effects of cisplatin and retinoic acid (RA) on HL-60 cells: pretreatment of HL-60 cells with 10(-7) M bufalin for 6 h increased the inhibitory effects of cisplatin and RA on cell growth and enhanced the induction of cell death.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently identified member of the secretin/vasoactive intestinal polypeptide (VIP) family. There are at least two types of receptor for PACAP: type I (PACAPR), which specifically binds PACAP; and type II (VIP/PACAPR), which binds both PACAP and VIP. The localization of PACAPR in the rat brain was determined by in situ hybridization and immunocytochemistry. We raised antisera against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR in order to localize PACAPR-like immunoreactivity (PACAPR-LI) in the rat brain. In general, the distribution of PACAPR-LI correlated well with the distribution of PACAPR transcripts. Particularly strong PACAPR mRNA expression was detected in the olfactory bulb, hippocampus, cerebellum and hypothalamus and moderate labeling was detected in other scattered regions. At the cellular level, PACAPR-LI appeared to be concentrated predominantly in neuronal perikarya and dendrites. At the ultrastructural level, strong immunostaining for the PACAPR was found in plasma membranes, rough endoplasmic reticulum, cytoplasmic matrix, and at synapses. This study provides the basis for a better understanding of the functions of PACAP in the rat brain.

It was demonstrated recently that geranylgeraniol (GGO) has potent apoptosis-inducing activity in various lines of tumor cells, including HL-60 cells. In the present study, we found that GGO markedly inhibited the expression of a calcium-binding protein, calreticulin, prior to the induction of apoptosis in HL-60 cells. Furthermore, we also observed a significant decrease in the tyrosine phosphorylation of a 36-kDa protein that is a major tyrosine-phosphorylated protein in HL-60 cells. These findings suggest that decreases in levels of calreticulin and in the tyrosine phosphorylation of the 36-kDa protein might be associated with the induction of apoptosis by GGO in HL-60 cells.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.

Shimizu and Tsuji established a method of preparing colloidal platinum nanoparticles, whose average size is 2 nm, by ethanol reduction of H(2)PtCl(6) in the absence of protective agents for the particles. Platinum nanoparticles have negative surface potential and are stably suspended from an electric repulsion between them. The platinum nanoparticles reduced hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) below 0.1 ppm. It is necessary to use higher concentration of platinum nanoparticles for the reduction of 2,6-dichlorophenol indophenol (DCIP) than that of hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical, because reoxidation of DCIPH(2) (reduced) by oxygen was not negligible under our experimental conditions. These results indicate that electrons on platinum nanoparticles produced by the method of Shimizu and Tsuji can reduce hydrogen peroxide, DPPH radical or DCIP transferring electrons.

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chan'su, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

When myeloblastic ML1 cells were cultured in the presence of Vitamin K 2 (menaquinone, VK 2 ), the population of cells capable of reducing NBT increased to 83.5% at low VK 2 concentration of 1 μM, indicating VK 2 induces cellular differentiation. VK 2 also exerted differentiation-inducing action on histiocytic U937 and promyelocytic HL60 cell lines. None of these effects were observed with Vitamin K 1 (phylloquinone, VK 1 ), suggesting the geranylgeranyl group of the side chain of VK 2 to be essential to these effects. Combinations of VK 2 with other differentiation-inducers such as interferon-γ retinoic acid, or camptothecin additively or synergistically induced the differentiation of HL-60 cells. These results suggest that VK 2 may safely be used in differentiation therapy in combination with other inducers.

Distribution and ultrastructural localization of a novel phosphoneuroprotein with a molecular mass of 14 kDa (PNP 14), and expression of its mRNA were studied in the adult rat central nervous system (CNS) by immunocytochemistry and in situ hybridization histochemistry. PNP 14 immunoreactivity was abundant in the molecular layer, present moderately in the granular layer, and rare in the Purkinje cell layer of the cerebellar cortex. No PNP 14 immunoreactivity was detected in the cerebellar medulla. In the forebrain, immunoreactivity was found in the hippocampus, striatum, and throughout the cerebral cortex, especially in layer V. Electron microscopic immunocytochemical observation in the cerebellar cortex revealed many PNP 14-immunoreactive axon terminals making synaptic contact with dendritic processes in both granular and molecular layers. PNP 14 immunoreactivity was present mainly in the cytoplasmic matrix in the presynaptic axon terminals. PNP 14 mRNA was localized in the granular layer of the cerebellar cortex, in the hippocampus, and in the cerebral cortex, suggesting that PNP 14 is synthesized in neurons of the granular layer and then transported to the molecular layer by axonal transport. These morphological findings suggest that PNP 14 is likely to modulate the function of selected CNS synapses.

Pituitary adenylate cyclase-activating-polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide family of peptides; it occurs as two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. Rabbit antisera against synthetic PACAP27 were characterized by enzyme-linked immunosorbent assay. One of the antisera, using a high antibody titer, recognized both PACAP27 and PACAP38 and was found useful for immunohistochemistry. The distribution and ultrastructural localization of PACAP-like immunoreactivity (PACAP-LI) in the rat testes at different stages of spermatogenesis were studied with this antiserum. Four oligonucleotide probes, each complementary to a different region covering a different intron-exon junction, were chosen to maximize hybridization based on the predicted secondary structure of PACAP messenger RNA. PACAP-LI was detected in the developing germ cells but not in either Sertoli or Leydig cells. Intense PACAP-LI was found in spermatids situated near the lumen of the seminiferous tubules. Lower levels of PACAP-LI were detected in spermatogonia and primary spermatocytes, but no PACAP-LI was found in mature spermatids, testicular spermatozoa, or epididymal spermatozoa. In spermatids, PACAP-LI was detected during the cap phase and acrosome phase but not in the maturation phase. At the ultrastructural level, numerous gold particles representing PACAP-LI were found in both acrosomal granules and acrosomal caps of spermatids, while a few particles were found in the Golgi complex. Very few gold particles were seen in the acrosome of mature spermatids and spermatozoa. PACAP-LI decreased and finally disappeared from spermatids during the late developmental stages. In situ hybridization indicated that most of the signal was detected near the perimeter of seminiferous tubules in early developing germ cells, especially in spermatogonia and primary spermatocytes, suggesting that transcription of the PACAP gene occurs in spermatogonia and primary spermatocytes. The processing of the prohormone appears to be slow, and mature PACAP only appears in spermatids. These morphological findings suggest that PACAP-like substances, synthesized by germ cells, participate in spermatogenesis, particularly spermiogenesis, probably by an autocrine and paracrine mechanism. However, the possibility that PACAP acts on the Sertoli and/or Leydig cells cannot be excluded.

We found that bufalin, an active principle of the Chinese medicine chan'su, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth-inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL-60, ML1, and U937 leukemia cells treated with bufalin at 10(-8) M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL-60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL-60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL-60 cells was 10(-8) M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all-trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10(-6) M bufalin for 24 h. These results indicate the association of the growth-inhibitory effect of bufalin with the induction of apoptosis, at least in HL-60 cells, and suggest the usefulness of bufalin for differentiation-apoptosis-inducing therapy for cancer.

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and it's amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.

We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05–20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin.

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Inhibitors of angiotensin-converting enzyme, renin and angiotensin II receptor lower the blood pressure of spontaneously hypertensive rats (SHRs) used as a model of essential hypertension. Since their plasma renin levels were normal or subnormal, renin in the vascular tissue was considered to play a key role in the maintenance of the hypertension. To clarify the source and localization of vascular renin in SHRs, the effects on blood pressure of antirenin antibodies, the converting enzyme inhibitors delapril and enalapril, and the angiotensin II receptor antagonist DuP 753 were examined in intact and bilaterally nephrectomized SHRs and their normotensive controls. The efficient hypotensive action of the renin antibody indicated that renin of the renal origin is a dominant factor. The gradual but complete disappearance of the antihypertensive action of these inhibitors of the renin-angiotensin system upon bilateral nephrectomy indicated the importance of membrane-associated renin of the renal origin and angiotensin-converting enzyme in the maintenance of the spontaneous hypertension.

Inhibitors of angiotensin converting enzyme, renin, and the angiotensin II (Ang II) receptor lower the blood pressure of spontaneously hypertensive rats (SHR) used as a model of essential hypertension. Since their plasma renin levels were normal or subnormal, renin in the vascular tissue was considered to play a key role in the maintenance of the hypertension. To clarify the source and localization of renin in SHR, antirenin antibodies, the converting enzyme inhibitors delapril, enalapril, and the Ang II receptor antagonist DuP 753 were administered to intact and bilaterally nephrectomized SHR and their normotensive controls. The efficient hypotensive action of the renin antibody indicated that renin of renal origin is a dominant factor. Gradual but complete disappearance of antihypertensive action of these inhibitors of the renin-angiotensin system upon bilateral nephrectomy indicated the importance of membrane-associated renin of the renal origin and angiotensin converting enzyme in the maintenance of the spontaneous hypertension.

A new acidic protein specifically present in the brain was purified to homogeneity from bovine brain. The apparent molecular mass was estimated to be 14 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 57 kDa by gel filtration, a finding suggesting that it exists as a tetramer under physiological conditions. The protein had a high content of Glu and Pro, and its pI was 4.3. The first six amino acid residues of the protein were Met-Asp-Val-Phe-Met-Lys, and the amino terminal was blocked. The distribution of the protein examined by Ouchterlony gel immunodiffusion indicates that it is present specifically in brain, including rat, human, and bovine, but could not be detected in 10 other rat tissues examined. The protein was absent in Purkinje cell bodies, as examined by electron microscopic immunocyto-chemistry, but was present in nerve terminals that make synapse-like contacts with Purkinje cells and in neurons with dark granules in the globus pallidus of the rat.

Little is known about the native form of endothelin receptor. To clarify its functional and structural properties, we solubilized the receptor from human placenta in an active form using mild detergents CHAPS and digitonin and showed that it is able to bind 125I-endothelin-1 in a specific manner, with a pH optimum between 6 and 8 in contrast to a reported pH optimum of 4. The molecular weight of the receptor was estimated as 340,000 by gel filtration of the solubilized membrane in the presence of 0.2% ( w v ) digitonin. When the solubilized membranes were labeled with 125I-endothelin-1 prior to gel filtration, the radioactive ligand also migrated in the position corresponding to a 340 kDa protein. These results indicated that the native form of endothelin receptor in human placenta is a 340 kDa protein.