Sample collection from asteroid (162173) Ryugu by Hayabusa2: Implications for surface evolution.

The near-Earth asteroid (162173) Ryugu is thought to be a primitive carbonaceous object that contains hydrated minerals and organic molecules. We report sample collection from Ryugu's surface by the Hayabusa2 spacecraft on 21 February 2019. Touchdown images and global observations of surface colors are used to investigate the stratigraphy of the surface around the sample location and across Ryugu. Latitudinal color variations suggest the reddening of exposed surface material by solar heating and/or space weathering. Immediately after touchdown, Hayabusa2's thrusters disturbed dark, fine grains that originate from the redder materials. The stratigraphic relationship between identified craters and the redder material indicates that surface reddening occurred over a short period of time. We suggest that Ryugu previously experienced an orbital excursion near the Sun.

Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling.

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Characterization of the carboxyl terminal-truncated endothelin B receptor coexpressed with G protein-coupled receptor kinase 2.

The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressing the wild type or delta 40 ETBR, ET-1 caused rapid desensitization of calcium responses. The wild type ETBR was phosphorylated by biotinylated ET-1, and the phosphorylation was markedly enhanced by coexpression with G protein-coupled receptor kinase 2 (GRK2). However, delta 40 ETBR was not phosphorylated regardless of coexpression with GRK2. On the other hand, ET-1-induced IP3 formation in these cells was decreased by coexpression with GRK2 or catalytically inactive Lys220Arg GRK2 to the similar extent. The present study demonstrates the presence of phosphorylation-independent desensitization mechanism in delta 40 ETBR and suggests that GRK2 might play a role other than that as a kinase.

Novel mutations of the endothelin B receptor gene in patients with Hirschsprung's disease and their characterization.

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.

Characterization of [3H]5-hydroxytryptamine and [3H]spiperone binding sites in clathrin-coated vesicles from bovine brain.

Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.

[Anesthesia for a patient with heparin-induced thrombocytopenia: a case report].

A 68 year-old man underwent a femoral-popliteal bypass surgery. He was diagnosed as heparin-induced thrombocytopenia (HIT) by platelet aggregation test when he underwent CABG 4 years ago. For the present surgery we administered nafamostat mesilate and urokinase for anticoagulation during surgery instead of heparin. After the operation, laboratory studies showed no thrombocytopenia.

Analysis of two pharmacologically predicted endothelin B receptor subtypes by using the endothelin B receptor gene knockout mouse.

1. This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETH receptor agonists in normal mice are the product(s) of a single ETB receptor gene. 2. Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET-1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ-123, or an ETA/ETB receptor antagonist, PD142893. 3. In the normal mouse aorta precontracted with phenylephrine, S6c (0.1-100 nM) caused concentration-dependent relaxations (pD2 = 8.4). BQ-123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1-300 nM S6c. 4. In aortae taken from ETB receptor gene knockout mice, S6c up to 1 microM failed to cause relaxations, confirming that ETB receptors are involved in mediating this response. 5. In normal mouse gastric fundus, 0.1 nM-1 microM ET-1, S6c or IRL1620 caused dose-dependent, BQ-123-insensitive contractions, which were much more resistant to PD142893 than S6c-induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 microM) were 8.12 +/- 0.11 and 7.70 +/- 0.11, respectively. 6. In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET-1 (0.1 nM-1 microM) caused contractions sensitive to both BQ-123 and PD142893, indicating that only ETA receptors mediate ET-1-induced contractions of the knockout mouse gastric fundus. 7. Since both the PD142893-sensitive vasodilator response of the aorta and the PD142893-resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

Expression of dopamine transporter at the tips of growing neurites of PC12 cells.

The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.

[The effects of preoperative drinking and H2 blocker on gastric acid secretion].

We studied the effects of preoperative drinking and H2 blocker on gastric acid secretion in 63 patients (ASA I-II, > 18yrs) scheduled for afternoon surgery. Group A (n = 20), as a control, was not permitted to eat and drink from 9 pm, the day before surgery, and was then given 500 ml of maintainance fluid before anesthesia. Group B (n = 20) fasted from 9 pm the day before surgery, and was allowed to drink clear fluids until 2hs before anesthesia. Group C (n - 23) followed the same guidelines as group B, and was given famotidine (20mg) orally at 9 pm the day before and 2hs before anesthesia. After induction, a Salem sump tube was inserted into the stomach and a gastric fluid aspiration was performed. The fluid volume and pH were measured after collection. Gastric pH was significantly higher (P < 0.001) in group C (6.4 +/- 0.9) than in groups A (3.1 +/- 1.8) and B (2.7 +/- 1.8). Fluid volume was similar in each group (A; 11 +/- 9/B; 12 +/- 9/C; 12 +/- 13ml). The dilution of gastric acid by the ingested fluid was not observed. We conclude that preoperative drinking does not affect gastric contents in elective operative patients. To reduce the risk of developing aspiration pneumonia, we recommend that every patient should receive an oral H2 blocker.

Endothelin ETA and ETB receptors mediate endothelin-1-induced apamin-sensitive relaxation in the guinea pig ileum.

Endothelin (ET) receptors involved in ET-1-induced responses of the longitudinal muscle of the isolated guinea pig ileum were studied. ET-1 caused concentration-dependent contractions, while ET-3 and selective ETB-receptor agonists, IRL1620 and sarafotoxin 6c (S6c), showed little or no effect. The ET-1-induced contractions were antagonized by BQ-123, an ETA-receptor antagonist, or PD142893, an ETA/ETB-receptor antagonist, indicating that the contraction is mediated by the ETA receptor. In preparations precontracted with carbachol, ET-1 elicited relaxations at lower concentrations and contractions at higher concentrations. ET-3, IRL1620 and S6c caused relaxations. These relaxations were little affected by BQ-123, but greatly antagonized by PD142893. The ET-1-induced relaxations were slightly affected by BQ-788, an ETB-receptor antagonist, but were markedly inhibited by the combination of BQ-788 and BQ-123. In ETB receptor-desensitized preparations, ET-1-induced relaxations were antagonized by BQ-123, whereas ET-3, S6c and IRL1620 showed no response. All these relaxations were abolished by apamin. These results indicate that ETA and ETB receptors mediate relaxation of the ileal smooth muscle through activation of apamin-sensitive K+ channels.

Heterogeneity of endothelin ETA receptor-mediated contractions in the rabbit saphenous vein.

The involvement of endothelin ETA receptors in endothelin-1-induced contractions of the rabbit saphenous vein was studied. After desensitization of endothelin ETB receptors by pretreatment with sarafotoxin S6c, endothelin-1 and sarafotoxin S6b and a high concentration of endothelin-3 caused dose-dependent contractions. However, endothelin-1-induced contractions were much less sensitive to an endothelin ETA receptor antagonist, BQ-123 (cyclo (-D-Asp-L-Pro-D-Val-L-Leu-D-Trp-)), than sarafotoxin S6b-induced responses. The pA2 values of BQ-123 for endothelin-1- and sarafotoxin S6b-induced contractions were 5.69 and 7.65, respectively. These results suggest pharmacological heterogeneity of endothelin ETA receptors in the rabbit saphenous vein.

Two different endothelin B receptor subtypes mediate contraction of the rabbit saphenous vein.

To study endothelin receptor subtypes that mediate venous smooth muscle contraction, effects of some endothelin receptor agonists and antagonists on the rabbit lateral saphenous vein were examined and compared with those on the saphenous artery. In the artery, endothelin (ET)-1 elicited concentration-dependent contractions, while selective ETB-receptor agonists, IRL1620 (Suc-[Glu9,Ala11,15]ET-1(8-21)) and sarafotoxin 6c (S6c) had almost no effect. The ET-1-induced responses shifted in parallel to the right by BQ-123 (cyclo (-D-Trp-D-Asp-Pro-D-Val-Leu-)), an ETA-receptor antagonist, or PD142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), an ETA/ETB-receptor antagonist, indicating the involvement of the ETA receptor in this response. In the saphenous vein, not only ET-1 and ET-3, but also ETB-receptor agonists, IRL1620, S6c and [Glu9]sarafotoxin 6b ([Glu9]S6b), produced concentration-dependent, BQ-123-insensitive contractions. PD142893 did not affect the ET-1-induced contraction, but it shifted greatly the IRL1620-induced concentration-response curve in parallel to the right. The major components of ET-3-, S6c- and [Glu9]S6b-induced contractions were resistant to PD142893. These results indicate that two different vasoconstrictive ETB-receptor subtypes, ETB1 (sensitive to IRL1620 and PD142893) and ETB2 (insensitive to IRL1620 and PD142893), are located on the smooth muscle of the saphenous vein.

Expression of a dopamine transporter in PC12h cells: an immunohistochemical study with an antipeptide antibody.

Two kinds of oligopeptides, based on the amino acid sequences of No. 217-232 and 374-387 of a rat dopamine transporter, were designed and chemically synthesized. Five clones of the monoclonal antibodies against these peptides were produced with the in vitro immunization method. Two of them have recognized a protein of M(r) approximately 85,000 in a synaptosomal fraction of the rat striatum. It is probable that the protein M(r) approximately 85,000 corresponds to a dopamine transporter in the nerve terminal of the dopamine neurons in the striatum. The expression of dopamine transporter was immunohistochemically examined with these antibodies in PC12h cells. The immunoreactivity was detected on the surface membrane of the cells.

Cloning and sequencing of a human endothelin converting enzyme in renal adenocarcinoma (ACHN) cells producing endothelin-2.

Endothelin (ET)-2 is a 21 residue vasoactive peptide which is biosynthesized from big ET-2(1-38) by a specific cleavage at Trp21-Val22 with an ET converting enzyme (ECE). To identify an ECE in ACHN (human renal adenocarcinoma) cells which produce ET-2, we have cloned and sequenced a novel cDNA encoding a human ECE in ACHN (hAECE). It encodes a 770 amino acid protein with a zinc-binding motif and a single membrane spanning region. The sequences of nucleic acids and amino acids from Leu45 to Trp770 of hAECE are identical to those from Leu33 to Trp758 of a human ECE in HUVEC (hHECE). The sequences in the amino-terminal moiety are divergent between hAECE and hHECE. Based on the difference of the amino-terminal amino acid sequences, ECEs reported so far, can be classified into two isoforms. These results strongly suggest that an alternative splicing might occur in the 5'-terminal region of the ECE pre-mRNA.

Dopamine D1 and D2 receptors and their signal system present in coated vesicles prepared from bovine striatal tissue.

Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D1 and D2 receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D1 receptor antagonist, [3H]SCH 23390, and a dopamine D2 receptor antagonist, [3H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant (KD) of 0.65 and 0.5 nM, respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [3H]SCH 23390 and [3H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D1 or D2 receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [32P]NAD demonstrated predominant labeling of bands of M(r) 47,000-52,000, 42,000-45,000, and 40,000-39,000, which corresponded to the known molecular weights of the alpha subunits of Gs and Gi proteins. The presence of alpha and beta subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D2 receptor agonists, was present in the CVs. These findings suggest that the dopamine D1 and D2 receptors in the CVs couple with adenylate cyclase via Gs/Gi protein.

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglion in vivo following stimulation-induced exhaustion.

Cat superior cervical ganglia (SCG) were tetanically stimulated in vivo at 30-100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 microns-2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 microns-2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.

Endothelin-2-converting enzyme from human renal adenocarcinoma cells is a phosphoramidon-sensitive, membrane-bound metalloprotease.

From the membrane fraction of cultured human renal adenocarcinoma (ACHN) cells, two endothelin-2-converting enzymes (ECE-2A and ECE-2B) were solubilized with detergent Lubrol PX and separated by hydrophobic butyl fast-performance liquid chromatography. The pH range of the converting activity of ECE-2B for big endothelin-1 (big ET-1), big ET-2, or big ET-3, was very narrow, and the optimal pH for each substrate was significantly different; the pH optimum for big ET-1 was 6.8 and that for big ET-2 or big ET-3 was 6.4. The ET-converting activity was abolished by phosphoramidon, 1,10-phenanthroline and EDTA but was not inhibited by thiorphan, E-64, leupeptin, PCMS, p-APMSF, or pepstatin A. The conversion efficiency for big ET-2 or big ET-3 by ECE-2B was approximately one-eighth of that for big ET-1. The molecular weight of ECE-2B was estimated to be 400 kDa by gel filtration. Because these characteristics of ECE-2B are very similar to those of ET-1-converting enzyme (ECE-1) in endothelial cells, these results raise the possibility that ECE-2B is identical to ECE-1 and that a single ECE physiologically converts all big ETs to the corresponding ETs in ET-producing cells.

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.

Localization of a 82 kDa protein in postsynaptic density and its association with cytoskeletons.

A fraction of synaptic junctional complex (SJC) was prepared from rat synaptosomes and served as antigen material to produce monoclonal antibodies (Mab) for examining the component proteins of the SJC. An antibody, Mab SJ-8, was obtained, which recognized a protein with a molecular weight of 82,000 Da in the SJC preparation by immunoblot analysis. The immunohistochemical localization of the 82 kDa protein was studied with the rat cerebellum. Mab SJ-8 labeled the peripheral areas of the Purkinje and granule cells. Small punctate areas were also stained in the molecular layer with SJ-8. Intracellular localization of the protein was examined with rat brain synaptosomes. Immunoelectron microscopy demonstrated that Mab SJ-8 strongly labeled the postsynaptic density (PSD) and also a fibrous network spreading out of it. However, the antibody did not label the pre- or post-synaptic membrane or the cleft material.