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Biomedical subjects

M Takano

Publications and source records attributed to M Takano.

At least 271 records · Page 15Linked to original sources
PubMed1994

p-Aminohippurate transport in apical and basolateral membranes of the OK kidney epithelial cells.

p-Aminohippurate (PAH) transport in apical and basolateral membranes of OK cells was studied to characterize each membrane process and to determine which process is important for the vectorial transport of PAH across the OK cell monolayers. PAH uptake from the basal side into OK cells was much higher than from the apical side, and was inhibited almost completely by anion transport inhibitors (probenecid and furosemide) and low temperature. On the other hand, PAH efflux from OK cells to the apical side was higher than to the basal side. The efflux across the apical membrane also was inhibited by the transport inhibitors and low temperature. The uptake of PAH across the basolateral membrane was inhibited by dicarboxylates such as alpha-ketoglutarate, and kinetic analysis showed that the inhibition was a mixed-type. These findings suggest that PAH transport in apical and basolateral membranes of OK cells is a specifically mediated process, and both processes contribute to the vectorial transport of PAH across the cell monolayers. PAH/dicarboxylate exchange may be involved in PAH transport in the basolateral membrane of OK cells.

Animals↗
PubMed1994

Transport of gentamicin and fluid-phase endocytosis markers in the LLC-PK1 kidney epithelial cell line.

In order to characterize the transport of aminoglycoside in the cultured kidney epithelial cell line LLC-PK1, cellular uptake of gentamicin was studied in comparison with those of fluid-phase endocytosis markers, lucifer yellow and horseradish peroxidase. The uptake of gentamicin and fluid-phase markers were time-dependent and were reduced at low temperature. The rate of gentamicin uptake was, however, faster than those of lucifer yellow and horseradish peroxidase, and was saturable. The accumulation of gentamicin in the cells was increasing even after 6 days of incubation, whereas that of lucifer yellow reached a steady state by 1 day. Release of intracellular gentamicin from LLC-PK1 cells was much slower than that of lucifer yellow. Gentamicin uptake, but not lucifer yellow uptake, was increased by reducing the ambient ionic strength. Under the low ionic strength conditions, gentamicin uptake was inhibited by the presence of other aminoglycosides, lysozyme, polyamines such as spermine and inorganic cations such as calcium. These results indicate that gentamicin electrostatically binds to the cell apical membrane and is subsequently taken up by an adsorptive endocytosis in LLC-PK1 cells.

Animals↗
PubMed1994

Fractional mean transit time in transplanted kidneys studied by technetium-99m-DTPA: comparison of clinical and biopsy findings.

UNLABELLED: To determine the usefulness of fractional mean transit time (MTT) in the differential diagnosis of postrenal transplant complications, 99mTc-DTPA was used to evaluate differences in MTT between the outer zone (cortical nephron) and middle zone (juxtamedullary nephron, calcyces and cortical nephron) of the kidney. It is well known that acute rejection is characterized by delayed cortical transit time, whereas cortical nephron function is well maintained and juxtamedullary function is impaired after renal ischemia. METHODS: Technetium-99m-DTPA fractional MTT was determined by deconvolution analysis of 89 renograms obtained within 5 days of the date of kidney graft biopsy and evaluation. RESULTS: Outer zone MTT was significantly shorter than middle zone MTT in normals (2.7 +/- 0.4 versus 3.0 +/- 0.6 min, n = 22, p < 0.001), acute tubular necrosis (3.4 +/- 1.1 versus 3.6 +/- 1.4 min, n = 19, p < 0.01), chronic rejection (3.9 +/- 1.5 versus 5.0 +/- 2.3 min, n = 14, p < 0.001) and obstruction (4.1 +/- 0.6 versus 8.9 +/- 3.4 min, n = 13, p < 0.001). In contrast, outer zone MTT was significantly longer than middle zone MTT in acute rejection (4.8 +/- 3.2 versus 4.2 +/- 2.5 min, n = 21, p < 0.05). CONCLUSION: Fractional MTT was demonstrated to be useful in differentiating acute rejection and ATN in transplanted kidneys.

Adolescent↗
PubMed1993

Transport of organic cation in renal brush-border membrane from rats with renal ischemic injury.

Transport of tetraethylammonium, an organic cation has been studied using renal brush-border membrane vesicles isolated from rats with ischemic and ischemia-reperfusion injury. H+ gradient-dependent uptake of tetraethylammonium slightly, but significantly, decreased in brush-border membrane vesicles from ischemic kidneys. When the kidney was reperfused after ischemia, the extent of the decrease of tetraethylammonium uptake was much greater than that after ischemia alone. The Vmax value of tetraethylammonium uptake by brush-border membrane vesicles from reperfused kidneys was decreased compared with control, without any change in the Km value. The tetraethylammonium uptake by the vesicles from reperfused kidneys was decreases both in the presence and absence of the outward H+ gradient (driving force). Uptake of D-glucose in renal brush-border membrane vesicles was also decreased by ischemia and again, reperfusion caused a further decrease of the uptake. Reperfusion also induced marked changes in the enrichment and recovery of marker enzymes in the isolated brush-border membrane fraction compared with ischemia. These findings suggest that renal ischemic injury altered the transport properties of tetraethylammonium as well as D-glucose, and that reperfusion after ischemia induced further damages on these functions in the brush-border membrane.

Animals↗
PubMed1993

Transport of bestatin in rat renal brush-border membrane vesicles.

Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] is a dipeptide, comprising L-leucine and an unusual beta-amino acid. We studied its transport mechanism in rat renal brush-border membrane vesicles. Uptake of cephradine, an aminocephalosporin, by isolated brush-border membrane vesicles was trans-stimulated and cis-inhibited by bestatin, indicating that these drugs are transported via the same transport system(s). The uptake of bestatin was trans-stimulated by preloading the vesicles with glycylsarcosine, and was cis-inhibited by substrates for the H+/dipeptide cotransport system. Bestatin inhibited tetraethylammonium (an organic cation) uptake, and bestatin uptake was cis-inhibited by substrates for the H+/organic cation antiport system. In addition, bestatin uptake was stimulated by an outward H+ gradient (the driving force for the H+/organic cation antiport system). These findings suggest that bestatin, in spite of being a dipeptide, is transported via not only the H+/dipeptide cotransport system but also the H+/organic cation antiport system in rat renal brush-border membrane.

Animals↗
PubMed1993

Characteristics of organic cation transporter in rat renal basolateral membrane.

Characteristics of organic cation transport system were studied in rat renal basolateral membrane and compared with those in brush-border membrane. We first examined the effect of various chemical modifiers on tetraethylammonium uptake by the membrane vesicles. Treatment with N,N'-dicyclohexylcarbodiimide and phenylglyoxal (carboxyl groups and arginine residues specific reagent, respectively) resulted in inhibition of tetraethylammonium transport in both basolateral and brush-border membranes. Tetraethylammonium uptake by brush-border, but not by basolateral, membrane vesicles was decreased by diethyl pyrocarbonate, histidine residues specific reagent, treatment. Treatment of sulfhydryl groups with HgCl2 decreased tetraethylammonium transport in both membranes. However, in contrast to brush-border membrane, unlabeled tetraethylammonium failed to protect against the inhibition of [14C]tetraethylammonium uptake by p-chloromercuribenzene sulfonate in basolateral membrane. We next examined the inhibitory effect of various organic cations on tetraethylammonium uptake. The order of inhibitory potency of organic cations was somewhat different between two membranes. These findings suggest that the characteristics of organic cation transport systems in basolateral and brush-border membranes were different in regard to essential amino acid residues and the affinity of substrates.

Amiloride↗
PubMed1993

Interleukin-6 as a mediator responsible for inflammation-induced increase in plasma angiotensinogen.

The concentration of plasma angiotensinogen increases upon induction of inflammation. Studies were carried out using serum samples collected from mice and rats after injection of lipopolysaccharide (LPS) to determine whether interleukin-6 (IL-6) is a mediator responsible for the inflammation-induced increase of angiotensinogen synthesis in liver cells. Serum collected from mice or rats 2 and 4 hr after injection of LPS contained a factor that stimulated [35S]methionine incorporation into angiotensinogen newly synthesized by rat hepatoma H4IIEC3 (H4) cells. Assay of IL-6 using an IL-6-dependent murine hybridoma, MH60.BSF2 cells, showed the presence of IL-6-like activity in sera of mice or rats 2 and 4 hr after injection of LPS. Anti-mouse IL-6 monoclonal antibody completely inhibited not only the IL-6-like activity present in LPS-treated mouse serum but also the ability of the serum to stimulate angiotensinogen synthesis of H4 cells. These results suggest that increased synthesis of angiotensinogen in the liver after induction of inflammation is mediated by IL-6, a cytokine important in immune reactions and the hepatic acute-phase response.

Angiotensinogen↗
PubMed1993

The ATP-sensitive K+ channel.

A small conductance K+ channel, that is inactivated by ATP, was recently found in the inner membrane of rat liver mitochondria (Inoue et al., 1991). This finding clearly indicates that a variety of K+ channels, showing ATP-sensitivity, are widely distributed. ATP is an important compound in view of its participation in oxidative phosphorylation and as the source of high-energy phosphate for nearly every energy-requiring reaction in the cell. Therefore, it is easy to speculate that transducing the ATP concentration within a cell into an electrical signal is vital for most living cells. The opening of the ATP-sensitive K+ channel by a decrease in the ATP level shifts the membrane potential in a negative direction and in general depresses cell function. The closing of the channel by an increase in ATP depolarizes the membrane and enhances membrane excitability. It might be speculated that a sequence of amino acids common for the binding site of ATP is preserved and combined with different types of K+ channels, so that the gating with ATP is quite similar between different K+ channels, but the conductance properties are different. The large variability in the value of K1/2ATP in the same cells or between different tissues might be due to modulation of the reaction of ATP and the binding site. These ideas will be substantiated by clarifying the molecular structure of the ATP-sensitive K+ channel in the near future. The molecular mechanisms for the selective channel blockers, sulfonylureas, and for the K+ channel openers should also be clarified.

Adenosine Triphosphate↗
PubMed1993

Decreased cellular toxicity of neomycin in a clonal cell line isolated from LLC-PK1.

We have previously shown in LLC-PK1 cells, that apical membrane enzyme activity was inhibited by aminoglycoside antibiotics (Am. J. Physiol. 254, C251-C257, 1988). In the present study, the relationship between the lethal cytotoxic effect of aminoglycoside and its effect on apical membrane enzyme was examined by establishing aminoglycoside resistant cells. A clonal cell line, LLC-PK1/NRa3, was isolated from parent LLC-PK1 cells in the presence of neomycin. Neomycin inhibited colony formation and increased the number of floating dead cells in parent LLC-PK1 cultures. In contrast, these cytotoxic effects of neomycin were negligible or less pronounced in NRa3 cells, indicating that NRa3 cells were more resistant to neomycin compared with the parent cells. The inhibitory effect of neomycin on apical enzyme activity was significantly weaker in NRa3 cells than in the parent cells. These results suggest that a common mechanism is involved in the aminoglycoside-induced reductions in the apical enzyme activity and in cell viability of LLC-PK1 cells.

Alkaline Phosphatase↗
PubMed1993

The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor derived from mammalian cells.

The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor expressed in CHO cells was investigated. The bioactive protein ([-32-153]M-CSF), expressed from a nucleotide sequence that encoded a signal peptide of 32 amino acids and N-terminal amino acids numbers 1-153, was heterogeneous in terms of molecular mass, as analyzed by SDS-PAGE, because of the presence of N-linked sugar moieties. The primary structure of the polypeptide was determined by sequence analysis and amino acid analysis of the fragments obtained from lysylendopeptidase digests of reduced and alkylated M-CSF, and from pepsin digests of the intact molecule. A sugar chain was located only at Asn-122 of the two putative sites of N-glycosylation that were present per subunit. The homodimeric structure appeared to have seven disulfide bonds, formed by inter- or intra-molecular linkages, since there were no free thiol groups in the molecule. The assignment of disulfide bonds by sequence analysis using peptide fragments indicated the combinations of Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146. Gel-filtration analysis of Ser31[-32-153]M-CSF, in which the remaining Cys31 was replaced by Ser and which was expressed in COS cells, suggested that the mutein existed as a monomer. Our study shows that the disulfide-bond pairings of [-32-153]M-CSF that is expressed and post-translationally modified in mammalian cells are identical to those of Escherichia coli-derived [3-153]M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.

Alkylation↗
PubMed1993

Transport of organic anion in the OK kidney epithelial cell line.

Transport of an organic anion, p-aminohippurate (PAH), was investigated in an established cell line derived from the American opossum kidney (OK). Transepithelial transport and accumulation of PAH were measured in OK cells cultured on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the transcellular transport of PAH from basal to apical side and the accumulation from basal side were temperature dependent, saturable, and inhibited by organic anions such as probenecid but not by organic cations. In contrast, such a specific transport of PAH was not observed in LLC-PK1, another epithelial cell line derived from pig kidney. These findings suggest that OK cells can transport PAH by the organic anion transport system unidirectionally from basal to apical side across the cell monolayers and that OK cells might be a useful model for the study of organic anion secretion in the renal proximal tubules.

Animals↗
PubMed1993

Elevation of plasma angiotensinogen in rats with experimentally induced nephrosis.

In order to determine the activity of the renin-angiotensin system in the nephrotic syndrome, the plasma concentration of angiotensinogen was measured in rats with puromycin aminonucleoside (PA)-induced nephrosis using two different methods: a direct radioimmunoassay, which measures both angiotensinogen and des-angiotensin I-angiotensinogen, and an indirect assay, which measures angiotensin I liberated from angiotensinogen by excess renin. The plasma concentration of angiotensinogen as measured by the direct assay increased before the appearance of PA-induced hypoproteinemia or proteinuria and subsequently decreased to normal levels simultaneously with the appearance of proteinuria. The indirect assay of angiotensinogen also demonstrated an increased concentration of plasma angiotensinogen before the development of nephrosis, but the level decreased to below normal after the appearance of proteinuria. Both plasma renin concentration and renin activity also increased simultaneously with the increase in plasma angiotensinogen. The difference between the concentrations of plasma angiotensinogen determined by these methods increased before and during the early phase of PA-induced nephrosis, suggesting the increased consumption of angiotensinogen by renin during this period. Measurement of plasma corticosterone and serum interleukin-6 revealed that these circulating factors were not involved in the elevation of plasma angiotensinogen in rats with PA-induced nephrosis. These results indicate that the renin-angiotensin system is activated before the appearance of PA-induced nephrotic syndrome.

Angiotensinogen↗
PubMed1993

Effect of prostaglandins on renal function in uninephrectomized humans.

We examined the effects of unilateral nephrectomy (UN) and administration of indomethacin in 15 healthy humans. Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), urine flow, sodium and potassium excretions, urinary excretion rate of prostaglandin E2 (PGE2) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) in the remaining kidney were all significantly (p < 0.01) increased 10 days after UN. Indomethacin administered at 75 mg/day for 3 days abolished the increase in GFR, ERPF and sodium excretion with reduced urinary excretion rate of PGE2 and 6-keto PGF1 alpha but not the increase in urine flow and potassium excretion. These findings suggest that renal prostaglandins may play a role in renal functional adaptation following UN.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

p-Aminohippurate transport in rat renal brush-border membranes: a potential-sensitive transport system and an anion exchanger.

Transport mechanisms of p-aminohippurate (PAH) were investigated in rat renal brush-border membrane vesicles. The uptake of PAH was stimulated by an inside-positive membrane potential created by K+ and valinomycin. This potential-stimulated uptake of PAH was inhibited by various anion transport inhibitors and was saturable. In addition, PAH uptake in the presence of valinomycin was linearly increased in proportion to log[K+]out/[K+]in. On one hand, PAH uptake was stimulated by [14C]PAH/PAH or [14C]PAH/Cl- exchange, and the [14C]PAH/PAH exchange was insensitive to the membrane potential. The uptake by the exchanger was also inhibited by anion transport inhibitors, but the potential-stimulated uptake of PAH was more sensitive to furosemide and 4,4'-diisothiocyano-2,2'-disulfonic stilbene. On the contrary, [14C]PAH/PAH exchange was more sensitive to urate than the potential-stimulated uptake of PAH. These findings indicate that PAH is transported by two distinct transport systems in rat renal brush-border membranes, a potential-sensitive transport system and an anion exchanger.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Transport mechanism of choline in rat renal brush-border membrane.

The transport mechanism of choline was examined using rat renal brush-border membrane vesicles in comparison with tetraethylammonium transport. The stimulatory effect of an outward H+ gradient on choline uptake was weak compared with that on tetraethylammonium uptake. [14C]Tetraethylammonium uptake was cis-inhibited and trans-stimulated by choline, but the effects were less pronounced than those produced by unlabeled tetraethylammonium. [3H]Choline uptake was trans-stimulated by unlabeled choline, but not by tetraethylammonium. An interior-negative membrane potential induced marked stimulation of choline uptake against its concentration gradient (overshoot phenomenon), and the uptake was saturable with an apparent Km of 0.77 mM. Various compounds such as hemicholinium-3 inhibited the choline uptake by renal brush-border membrane vesicles, but a sulfhydryl reagent did not. These findings suggest that choline can be actively transported by a carrier-mediated system driven by cell interior-negative membrane potential in renal brush-border membrane, and this system may play an important role in the tubular reabsorption of choline.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Endotoxin-induced enhancement of angiotensinogen synthesis in the liver: decreased response following repeated endotoxin exposure.

To understand the mechanisms responsible for lipopolysaccharide (LPS)-induced enhancement of angiotensinogen synthesis in the liver, studies were carried out in rats with repeated doses of LPS. The administration of sublethal dose (50 micrograms, i.p.) of LPS to rats resulted in increase in serum levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6), which attained their maximal levels by 1 and 2-4h, respectively. Serum levels of angiotensinogen and alpha 2-macroglobulin, a typical acute-phase protein in the rat, were also increased by a primary LPS challenge, and their maximal levels for the formation of TNF and IL-6 were delayed with peaks at 12 and 48 h, respectively. Repeated i.p. administration of LPS (50 micrograms/d) for 5 consecutive days induced a hyporesponsiveness to its subsequent administration in terms of increasing serum TNF, IL-6 and alpha 2-macroglobulin. In these LPS-tolerant rats, either LPS-induced elevation of angiotensinogen concentration in serum or angiotensinogen mRNA levels in liver was completely eliminated. Angiotensinogen synthesis in rat hepatoma H4 cells was enhanced in vitro by the addition of sera which had been collected 2 or 4 h after a primary injection of LPS, while not by sera collected from LPS-pretreated rats after a secondary LPS exposure. These results indicate that LPS-induced enhancement of angiotensinogen synthesis in the liver is desensitized in rats after repetitive LPS exposure, presumably by the failure of LPS-induced IL-6 production.

Angiotensinogen↗
PubMed1993

[Structure of adenylate cyclase and the coupling with the receptor-G protein system].

Adenylate cyclase is a key enzyme that couples with both the stimulatory and inhibitory G proteins (Gs and Gi). The cyclase has been purified and shown to be a glycoprotein of molecular weight 115,000-180,000. Cloning of cDNAs for adenylate cyclase showed that the cyclase is a member of a large family consisting of a variety of subtypes of the enzyme. These subtypes show different responses to calmodulin and G protein beta gamma subunits, and their distributions in tissues and organs are also different. This suggests that each subtype is involved in a particular physiological function. The general structure of adenylate cyclase is composed of two cytoplasmic domains and two membrane-spanning domains, each of which contains 6 transmembrane spans (12 spans in a molecule). The amino acid sequence of each cytoplasmic domain, which is thought to contain a nucleotide (ATP) binding site, is well-conserved among the various subtypes. This review also focuses on the regulation of adenylate cyclase activity by G protein subunits, particularly on several models for adenylate cyclase inhibition by Gi. As one of these mechanisms, direct inhibition of adenylate cyclase by the beta gamma subunits recently demonstrated by us will be discussed.

Adenylyl Cyclases↗