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Y Sai

Publications and source records attributed to Y Sai.

At least 37 records · Page 2Linked to original sources
PubMed2000

Limited distribution of new quinolone antibacterial agents into brain caused by multiple efflux transporters at the blood-brain barrier.

Transport of new quinolone antibacterial agents (quinolones) at the blood-brain barrier (BBB) was studied in vitro by using immortalized rat brain capillary endothelial cells RBEC1, and in vivo by using the brain perfusion method in rats and multidrug-resistant mdr1a/1b gene-deficient mice. The permeability coefficient of grepafloxacin measured by brain perfusion was increased by an excess of unlabeled grepafloxacin, suggesting a participation of a saturable BBB efflux system. Uptake coefficients of [(14)C]grepafloxacin, [(14)C]sparfloxacin, and [(14)C]levofloxacin by RBEC1 cells at the steady state were increased in the presence of the unlabeled quinolones. The steady-state uptake of [(14)C]grepafloxacin was increased in the presence of various quinolones. Brain distributions of [(14)C]grepafloxacin and [(14)C]sparfloxacin evaluated in terms of the brain-to-plasma free concentration ratio in mdr1a/1b gene-deficient mice were significantly higher than those in wild-type mice, demonstrating an involvement of P-glycoprotein as the efflux transporter. Anionic compounds, including 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and genistein, increased the steady-state uptake of [(14)C]grepafloxacin by RBEC1 cells. Because [(14)C]grepafloxacin was transported by multidrug resistance-associated protein (MRP), in MRP1-overexpressing cells and because RBEC1 and primary cultured brain capillary endothelial cells expressed MRP1, this protein may be an additional efflux transporter for quinolones. Furthermore, the permeability coefficient of [(14)C]grepafloxacin across the BBB was increased by DIDS or in the absence of bicarbonate ions in the brain perfusion method. DIDS or bicarbonate ion did not affect MRP1 function. Accordingly, the brain distribution of quinolones is restricted by the action of multiple efflux transporters, including P-glycoprotein, MRP1, and an unknown anion exchange transporter.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter.

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Amino Acid Sequence↗
PubMed1999

Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1.

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.

Amino Acid Sequence↗
PubMed1999

Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.

The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin. WIF-B9 cells were transiently and stably transfected with a mdr1-GFP construct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basolateral plasma membrane. At 15 degrees C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocytes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters. Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html

ATP-Binding Cassette Transporters↗
PubMed1999

Carrier-mediated lung distribution of HSR-903, a new quinolone antibacterial agent.

HSR-903 [(S)-(-)-5-amino-7-(7-amino-5-azaspiro[2. 4]hept-5-yl)-1-cyclopropyl-6-fluoro-1, 4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic acid methanesulfonate] is a newly synthesized quinolone with a potent antibacterial activity and a low toxicity. The lung concentration of unchanged HSR-903 was about nine times higher than that in plasma after oral administration (5 mg/kg) in rats. In comparative studies, HSR-903 was accumulated more efficiently than levofloxacin, ciprofloxacin, and lomefloxacin in rat lung. To clarify the mechanism of the specific distribution of HSR-903 into the lung, the uptake of [14C]HSR-903 was studied using isolated rat lung cells and an isolated rat lung perfusion technique. Initial uptake of HSR-903 by isolated lung cells was temperature dependent, saturable, stereospecific, and Na+ and Cl- dependent. The Hill coefficients (1. 90 for Na+ and 1.13 for Cl-) suggest that two Na+ and one Cl- are associated with the transport of one HSR-903 molecule. The uptake of HSR-903 was inhibited by other quinolone antibacterial agents, grepafloxacin, and sparfloxacin. The extraction ratio of HSR-903 in isolated lung perfusion was temperature dependent and saturable. These findings suggest that HSR-903 is taken up by the lung cells via a carrier-mediated transport mechanism, resulting in a concentrative distribution into the lung.

Algorithms↗
PubMed1999

Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations.

In the present study, functional characteristics of organic cation transporter (OCTN)1, which was cloned as the pH-dependent tetraethylammonium (TEA) transporter when expressed in mammalian human embryonic kidney (HEK)293 cells, were further investigated using Xenopus oocytes as well as HEK293 cells as gene expression systems. When OCTN1-derived complementary RNA was injected into Xenopus oocytes, pH-dependent transport of [14C]TEA was observed as the same in HEK293 cells. In contrast, a replacement of sodium ions with potassium ions in the surrounding medium did not cause any change in [14C]TEA uptake in Xenopus oocytes expressed with OCTN1. In addition, when OCTN1 was expressed in HEK293 cells, efflux of TEA from the cells was pH dependent, with an accelerated rate at acidic external medium pH. Accordingly, membrane potential or sodium ions are suggested to have no influence on [14C]TEA transport and the transport activity of OCTN1 is directly affected by pH itself. Furthermore, addition of the unlabeled TEA in external medium enhanced the efflux of preloaded [14C]TEA. These observations suggest that OCTN1 is a pH-dependent and bidirectional TEA transporter. OCTN1-mediated [14C]TEA uptake was inhibited by various organic cations such as cimetidine, procainamide, pyrilamine, quinidine, quinine, and verapamil. In addition, uptakes of cationic compounds such as [3H]pyrilamine, [3H]quinidine, and [3H]verapamil and zwitterionic L-[3H]carnitine were increased by expression of OCTN1 in Xenopus oocytes. Accordingly, OCTN1 was functionally demonstrated to be a multispecific and pH-dependent organic cation transporter, which presumably functions as a proton/organic cation antiporter at the renal apical membrane and other tissues.

Animals↗
PubMed1999

An inhibitory monoclonal antibody to human cytochrome P450 2A6 defines its role in the metabolism of coumarin, 7-ethoxycoumarin and 4-nitroanisole in human liver.

Cytochrome P450 (CYP) 2A6 is an important enzyme catalysing the metabolism of many drugs, procarcinogens and promutagens. Its role in human liver metabolism of coumarin, 4-nitroanisole, 4-nitrophenol and 7-ethoxycoumarin was analysed with an inhibitory monoclonal antibody (MAb) to CYP2A6. MAbs were derived from a panel of 16 hybridomas which yielded positive enzyme-linked immunosorbent assay (ELISA) results or immunoblots against CYP2A6. The hybridomas were selected from more than 500 clones generated by the fusion of myeloma cells with spleen cells of mice immunized with purified baculovirus-expressed human CYP2A6. The MAbs obtained from four of the 16 hybridomas exhibited strong inhibitory activity to CYP2A6-catalysed phenanthrene metabolism. MAb 151-45-4 was positive and highly specific to CYP2A6 as determined by ELISA and immunoblot, and showed no cross-reactivity with recombinant human CYP 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, as tested with ELISA and immunoblot analyses. MAb 151-45-4 specifically inhibited CYP2A6-catalysed metabolism of phenanthrene, 4-nitroanisole, 4-nitrophenol, coumarin and 7-ethoxycoumarin each by 94-99% and did not inhibit their metabolism catalysed by 10 other human CYPs. The potent inhibitory effect of MAb 151-45-4 was used to define the contribution of human CYP2A6 to the metabolism of coumarin, 4-nitroanisole and 7-ethoxycoumarin in seven human liver microsome samples. Coumarin metabolism in all of the seven samples was inhibited by greater than 94% by MAb 151-45-4 which indicates that essentially all microsome mediated coumarin metabolism in human liver is catalysed only by CYP2A6. Inhibition of 4-nitroanisole and 7-ethoxycoumarin metabolism by anti 2A6 MAb ranged from 22-65% and 8-24%, respectively. The degree of inhibition defines the contribution of CYP2A6 activity to the 4-nitroanisole and 7-ethoxycoumarin metabolism in human liver and the range reflects the variability among samples. The inhibitory antibody to CYP2E1 was used to determine its role in 4-nitroanisole and 7-ethoxycoumarin metabolism in seven human liver samples. The addition of both MAbs to CYP2A6 and 2E1 to the microsome samples defined combinatorially the relative role of CYP2A6 and 2E1 in the metabolism of 4-nitroanisole and 7-ethoxycoumarin.

Animals↗
PubMed1999

Na(+)-dependent carnitine transport by organic cation transporter (OCTN2): its pharmacological and toxicological relevance.

Carnitine deficiency, either primary or drug-induced, causes critical symptoms and is thought to involve alteration of active transport of carnitine across the plasma membrane of tissues as the underlying mechanism. Recently, we showed that human organic cation transporter, hOCTN2, cloned as a member of the organic cation transporter family, is a physiologically important Na(+)-dependent high-affinity carnitine transporter in humans. In this study, we further characterized the functional properties of hOCTN2 and examined the interaction between hOCTN2-mediated carnitine transport and clinically used drugs to assess possible toxicological effects. When expressed in human embryonic kidney (HEK)293 cells, hOCTN2 showed low but significant stereospecific transport activity: D-carnitine was transported with lower affinity (K(m) = 10.9 microM) than the L-isomer (K(m) = 4.3 microM). One Na(+) appeared to be associated with the transport of one carnitine molecule. hOCTN2-mediated transport of acetyl-L-carnitine was also Na(+)-dependent and of high affinity, with a K(m) value of 8.5 microM. To examine the transport activity for organic cations other than carnitine and the possible relationship of drug-induced carnitine deficiency with hOCTN2, the inhibitory effect of several drugs on hOCTN2-mediated L-carnitine transport was examined. Many zwitterionic drugs, such as cephaloridine, and many cationic drugs, such as quinidine and verapamil, exhibited significant inhibitory effects. Among these inhibitors, tetraethylammonium, pyrilamine, quinidine, verapamil, and valproate were found to be transported by hOCTN2. The results suggest that the carnitine deficiency-related toxicological effects by long-term treatment with such drugs might be ascribed to a functional alteration of hOCTN2-mediated carnitine transport.

Biological Transport, Active↗
PubMed1999

Eight inhibitory monoclonal antibodies define the role of individual P-450s in human liver microsomal diazepam, 7-ethoxycoumarin, and imipramine metabolism.

Eight inhibitory monoclonal antibodies (MAbs) individually specific to human cytochrome P-450 (P-450) 1A1, 1A2, 2A6, 2B6, 2C subfamily (2C8, 2C9, 2C18 and 2C19), 2D6, 2E1, and 3A4/5 were used to define the role of single P-450s in the metabolism of diazepam (DZ), 7-ethoxycoumarin (7-EC), and imipramine (IMI) in human liver microsomes (HLM). The MAbs were added combinatorially to six HLM samples. With DZ as a substrate, more than 80% of temazepam (TMZ) formation was inhibited in all six samples by the addition of MAb to 3A4/5, indicating an 80% contribution of 3A4/5 to TMZ formation. Nordiazepam formation was inhibited with MAbs to 2B6 (6-23%), 2C subfamily (12-61%) and 3A4/5 (14-45%). The MAbs to 1A1, 1A2, 2A6, 2D6, and 2E1 did not inhibit TMZ or nordiazepam formation; this indicates their noninvolvement in DZ metabolism. The MAb-defined P-450 contribution to 7-EC Odeethylation in six HLM samples was 17 to 60% for 2E1, 15 to 46% for 2A6, and 5 to 22% for 1A2, reflecting the role and variation of each P-450 in this activity. MAbs to 1A1, the 2C subfamily, 2D6, and 3A4/5 did not affect 7-EC metabolism in the HLM samples. IMI is metabolized mainly to 2-hydroxyimipramine by expressed 2C19 and 2D6, and desipramine (DIM) by expressed 1A2, 2C18, 2C19 and 2D6. Expressed 1A1, 2C9, and 3A4 showed low activities for the formation of DIM. Of six HLM samples, five showed IMI hydroxylation activity (0.35-2.6 nmol/min/nmol P-450) while one (HL43) lacked hydroxylation activity. All six HLM samples showed N-deethylation activity (0.74-1.4 nmol/min/nmol P-450). The MAb-determined contribution of 2D6 and 2C19 to 2-hydroxyimipramine formation ranged from 47 to 90% and from 0 to 49%, respectively, while HL43 did not show 2-hydroxylation. The role of P-450s involved in DIM formation varied for 2C19 (13-50%), 1A2 (23-41%), and 3A4 (8-26%). These studies demonstrate a system for identifying the quantitative metabolic role of single P-450s and their interindividual variability in a tissue containing multiple P-450s. The system using inhibitory MAbs is simple, precise, and applicable to any P-450-mediated catalytic activity including that for drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

Antibodies, Monoclonal↗
PubMed1998

Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2.

Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake of L-[3H]carnitine was strongly enhanced in a sodium-dependent manner with Km value of 4.34 microM, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediated L-[3H]carnitine transport was inhibited by the D-isomer, acetyl-D,L-carnitine, and gamma-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, beta-hydroxybutyric acid, gamma-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.

Acetylcarnitine↗
PubMed1998

Intestinal absorption of fluorescence-derivatized cationic peptide 001-C8-NBD via adsorptive-mediated transcytosis.

The intestinal absorption of an intact oligopeptide was investigated in rats using a synthetic cationic peptide, 001-C8 (H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2). The peptide was coupled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) to prepare a fluorescence-labeled derivative 001-C8-NBD (H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH-NBD) for the purpose of quantification. The degradation half-life of 001-C8-NBD in jejunal homogenate (1 mg/mL) was 99.5 min, which was significantly longer than that of natural leucine enkephalin (1.14 min). The absorption of 001-C8-NBD was evaluated by the vascular-perfusion method. Intact 001-C8-NBD appeared in the blood time-dependently and the absorption volume at 30 min (2.75 +/- 0.14 microL/cm intestine) was significantly larger than that of [3H]PEG 900 (0.88 +/- 0.13 microL/cm intestine), of which membrane permeability is very low. The absorption of 001-C8-NBD was greatly reduced by an adsorptive-mediated endocytosis inhibitor, protamine (10 mM). No inhibition of the absorption of [3H]PEG 900 by protamine was observed. The intestinal absorption was also measured by an in vivo loop method. The absorption clearance of 001-C8-NBD measured by this method (0.083 +/- 0.008 microL/min/cm intestine) was comparable to that obtained by the vascular perfusion method (0.092 +/- 0.005 microL/min/cm intestine). All of these data suggested that 001-C8-NBD was absorbed as the intact oligopeptide in the intestine in vivo. Adsorptive-mediated transcytosis is suggested to have enormous potential as an oral delivery system for peptide and/or protein drugs.

Animals↗
PubMed1998

Improvement of L-dopa absorption by dipeptidyl derivation, utilizing peptide transporter PepT1.

In the present study, possible enhancement of intestinal absorption of L-dopa by utilizing intestinal peptide transporter was examined using Caco-2 cells and Xenopus oocytes expressing human peptide transporter (hPepT1). To see whether this peptide transporter could be utilized for the improvement of L-dopa absorption, we employed a dipeptide-mimetic derivative of L-dopa, L-dopa-L-Phe. L-Dopa-L-Phe inhibited the uptake of [14C]Gly-Sar, but not that of L-[3H]-dopa by Caco-2 cells. Uptake of L-dopa-L-Phe was increased by expression of hPepT1 in Xenopus oocytes. The appearance of L-dopa and its metabolite, dopamine, on the basolateral side of Caco-2 cells was significantly higher after addition of L-dopa-L-Phe than after that of L-dopa and was reduced by the presence of Gly-Sar on the apical side. These results indicate that the L-dopa-L-Phe is absorbed more efficiently than L-dopa and is taken up via the peptide transporter, but not via the amino acid transporter, demonstrating the possibility of targeting the peptide transporter as a means for improving intestinal absorption of peptide-like drugs.

Amino Acid Transport Systems↗
PubMed1998

Possible role of anion exchanger AE2 as the intestinal monocarboxylic acid/anion antiporter.

PURPOSE: The purpose of the present study was to investigate the transport of organic monocarboxylic acids mediated by the anion exchanger AE2, which has been already reported to be present at several tissue cell membranes, including intestinal brush border membrane in rabbit. METHODS: Membrane transport of organic monocarboxylic acids by AE2 was investigated by transient AE2-gene expression in HEK 293 cells and subsequent uptake studies by the cells. RESULTS: Functional transfection of AE2 was confirmed by the enhanced 36Cl- efflux from the cells. When preloaded with chloride anion. AE2-transfected cells demonstrated a significantly enhanced [14C]benzoic acid transport activity compared with mock-transfected cells. The AE2-mediated uptake was saturable with kinetic parameters of Km = 0.26 +/- 0.08 mM and Vmax = 6.14 +/- 0.52 nmol/mg protein/3 min and the uptake of [14C]benzoic acid was pH-dependent with a maximal uptake at pH 6.5. AE2-mediated [14C]benzoic acid uptake was inhibited by Cl-, HCO3-, and DIDS. AE2-transfected cells demonstrated significantly enhanced transport activity for nicotinic acid, propionic acid, butyric acid, and valproic acid as well as benzoic acid compared with mock-transfected cells. CONCLUSIONS: AE2 is functionally involved in the anion antiport for organic monocarboxylic acids as well as inorganic anions and is supposed to play a partial role in the intestinal transport of organic acids.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Anaesthetic management for caesarean section combined with removal of phaeochromocytoma.

In this case report, the anaesthetic management for a removal of phaeochromocytoma undertaken immediately following Caesarean section is described. A 32-year-old female patients was given epidural anaesthesia for Caesarean section, and thereafter, general anaesthesia for a resection of phaeochromocytoma. During surgery, phentolamine, nitroglycerine and prostaglandin E1 were electively administered to decrease blood pressure and heart rate. A live infant was delivered and the supra-adrenal tumour was excised successfully. The patient's post-operative recovery was uneventful.

Adrenal Gland Neoplasms↗
PubMed1998

Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases.

We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.

Animals↗
PubMed1998

ARF-induced lysosomal lysis in vitro.

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) disintegrated lysosomes in a dose-dependent manner, as detected as the release of preloaded fluorescein isothiocyanate-dextran. The effect of GTPgammaS was suppressed by GTP or GDP, indicating a role of a GTP binding protein (G-protein) in the lysis [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877]. Gel filtration of cytosol and GTP-ligand blotting showed that a small GTP-binding protein participated in the lysosomal lysis. We partially purified the G-protein from rat liver cytosol and identified it as ARF1. GTPgammaS-stimulated lysis was reconstituted with ARF1 purified from bovine brain cytosol or recombinant ARF1. ARF bound to lysosomal membranes depending upon GTPgammaS in a dose-dependent manner. These results suggest that the transfer of ARF from the cytosol to the lysosomal membrane is necessary for GTPgammaS-stimulated lysis of lysosomes.

ADP-Ribosylation Factor 1↗
PubMed1998

Comparison of the effects of pancuronium and vecuronium in canine coronary and renal arteries.

BACKGROUND: Pancuronium has sympathomimetic actions but does not change or lowers systemic blood pressure in some studies of anesthetized humans and dogs. The present study was done to determine the actions and mechanisms of action of pancuronium on coronary and renal arteries other than those as a sympathomimetic agent. METHODS: Helical strips of coronary and renal arteries from mongrel dogs were suspended in oxygenated, warmed Ringer-Locke solution, and changes in the isometric tension were recorded. In some strips, transmural electrical stimulation (5 Hz for 40 s) was applied to activate perivascular adrenergic nerves. RESULTS: Pancuronium (10[-7] to 10[-5] M) caused dose-dependent relaxation in coronary and renal arteries contracted with prostaglandin (PG) F2alpha, whereas no significant response was induced with vecuronium. The relaxation was endothelium independent and abolished by indomethacin or tranylcypromine, a PGI2 synthase inhibitor. Transmural electrical stimulation caused coronary arterial relaxation, which was augmented by pancuronium and vecuronium. Desipramine also increased the response, and additional potentiation of the response was not elicited by pancuronium and vecuronium. In renal arteries, electrical stimulation caused contraction, which was also augmented by pancuronium and vecuronium. With desipramine treatment, these muscle relaxants did not potentiate the response. Endothelium-dependent coronary arterial relaxation caused by bradykinin was not affected by pancuronium. CONCLUSIONS: Pancuronium-induced relaxations in canine coronary and renal arteries appear to be mediated by PGI2 released from subendothelial tissues. Potentiations by pancuronium and vecuronium of the response to adrenergic nerve stimulation are expected to be due to an inhibition of the norepinephrine uptake but not to facilitated release of the amine.

Animals↗