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Biomedical subjects

M Hanano

Publications and source records attributed to M Hanano.

At least 55 records · Page 3Linked to original sources
PubMed1992

Significance of binding to Na,K-ATPase in the tissue distribution of ouabain in guinea pigs.

Ouabain binds specifically to Na,K-ATPase on the plasma membrane and therefore serves to measure the tissue concentration of Na,K-ATPase. We examined the role of ouabain binding to Na,K-ATPase in its overall tissue distribution. The tissue-to-plasma concentration ratio (Kp,vivo) was defined in each tissue after intravenous administration of 3H-ouabain in guinea pigs, and specific binding of ouabain to Na,K-ATPase was measured in tissue homogenate to obtain the dissociation constant and binding capacity in each tissue. A predicted tissue-to-plasma concentration ratio (Kp,vitro) was calculated using the obtained binding parameters and the volume of extracellular space in each tissue. The absolute values of Kp,vitro were comparable to those of Kp,vivo, except in brain. Regression analysis showed that the specific binding capacity of Na,K-ATPase in each tissue is the main factor in the tissue variation of Kp,vivo. Therefore, the binding of ouabain to Na,K-ATPase plays a significant role in the tissue distribution of ouabain.

Algorithms↗
PubMed1992

Localization of binding sites for epidermal growth factor (EGF) in rat kidney: evidence for the existence of low affinity EGF binding sites on the brush border membrane.

We investigated the renal distribution of 125I-EGF in the filtering perfused rat kidney using an acid washing technique. Trichloroacetic acid-precipitable 125I-EGF radioactivity was eluted from both the renal vein and the urinary cannulae, the former regarded as representing the antiluminal, and the latter the luminal, cell surface bound 125I-radioactivity. The addition of excess unlabeled EGF (20 nM) to the perfusate completely inhibited the binding of 125I-EGF to the antiluminal membrane but did not inhibit that of 125I-EGF to the luminal membrane. On the other hand, the order of relative density of 125I-EGF binding sites in the in vivo kidney determined by autoradiography was cortex > inner medulla > outer medulla. After the i.v. administration of excess unlabeled EGF together with 125I-EGF, the renal uptake of 125I-EGF was inhibited completely in the inner medulla, but only by 50% in the cortex and outer medulla, suggesting the presence of nonsaturable luminal uptake of EGF in the cortex and outer medulla. After i.v. administration of 125I-EGF, a change in position of silver grains from the luminal cell surface membrane to the intracellular space was observed in the proximal convoluted tubules. In conclusion, in addition to the previously identified uptake mechanisms of circulating EGF through high-affinity binding sites on the antiluminal cell surface membrane, the reabsorption mechanism of filtered EGF through low-affinity binding sites on the luminal cell surface membrane was demonstrated. In vivo autoradiography showed the gradual internalization of EGF from the luminal cell surface membrane to the intracellular space of the proximal convoluted tubule.

Animals↗
PubMed1992

Correlation between the inhibitory effects of basic drugs on the uptake of cardiac glycosides and taurocholate by isolated rat hepatocytes.

The role of the multispecific bile acid transporter for cardiac glycoside uptake is still controversial. This study was designed to examine the inhibitory effects of basic drugs (verapamil, dipyridamole, nifedipine, chlorpromazine, disopyramide, quinidine, propranolol, and lidocaine) on taurocholate uptake by isolated rat hepatocytes and to compare these effects with inhibition of ouabain uptake. Sodium-dependent taurocholate uptake was significantly reduced, to 50-70% of the control value, by 50 microM verapamil, dipyridamole, and nifedipine. Sodium-independent taurocholate uptake was more extensively inhibited, to 20-40%, by these basic drugs. The inhibition of ouabain uptake correlated better with sodium-independent taurocholate uptake (gamma = 0.918) than with sodium-dependent taurocholate uptake (gamma = 0.714). Taurocholate competitively inhibited ouabain uptake in the absence of sodium. These results indicate that the cardiac glycoside transport system is similar to the sodium-independent taurocholate transport system.

Animals↗
PubMed1992

Localization of epidermal growth factor (EGF) binding sites on antiluminal plasma membrane of rat kidney: autoradiographic study using nonfiltering perfused rat kidney.

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of 125I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of 125I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of 125I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of 125I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the 125I-EGF binding sites. Binding of 125I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of 125I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of 125I-EGF perfusion, suggesting the internalization of 125I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of 125I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.

Animals↗
PubMed1992

Renal tubular handling of p-aminohippurate and epidermal growth factor (EGF) in filtering and nonfiltering perfused rat kidneys.

We examined the integrity of renal tubular function in filtering and nonfiltering isolated perfused rat kidneys by using p-amino-3H-hippurate (3H-PAH) and the multiple indicator dilution method with 14C-creatinine as a reference. The influx clearance (PSu,1) of unbound 3H-PAH was 0.37 and 0.38 ml/sec in the filtering and nonfiltering kidneys, respectively. The efflux rate constants were comparable between filtering and nonfiltering kidneys, while the sequestration rate constant in the filtering kidney was approximately three times larger than that in the nonfiltering kidney. These data suggest that the nonfiltering kidney maintains 3H-PAH transporting ability through the antiluminal plasma membrane. The renal handling of epidermal growth factor (EGF) by filtering and nonfiltering kidneys was compared. The ratio of the total uptake of tracer 125I-EGF over 20 min in the nonfiltering kidney to that in the filtering kidney was 0.8. This ratio was reduced to 0.2 when the kidneys were perfused with tracer 125I-EGF plus 20 nM EGF. Furthermore, the total uptake of tracer 125I-EGF in the nonfiltering kidney was reduced 20-fold in the presence of 20 nM unlabeled EGF. These findings suggest that the tubular uptake of tracer 125I-EGF by filtering kidney takes place mainly via the antiluminal plasma membrane and that this uptake is a saturable process.

Animals↗
PubMed1992

Fibrinolysis and fibrinogenolysis in patients with thrombotic disease.

In order to assess the fibrinolytic state in thrombotic disease, plasma levels of fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) were measured in 126 patients with a variety of thrombotic diseases. Mean plasma concentrations of both FbDP and FgDP were significantly elevated in patients with thrombotic disease as compared with healthy subjects. Plasma concentrations of FgDP were positively correlated with FbDP (r = 0.667, P less than 0.001). When analysed according to the disease categories, the magnitude of elevations of FbDP and FgDP was most prominent in venous thrombotic disease such as deep vein thrombosis and pulmonary embolism. These findings indicate that fibrinolysis is accelerated in patients with thrombotic disease and that fibrinolysis is frequently accompanied by some fibrinogenolysis in these patients.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Importance of the liver in plasma clearance of hepatocyte growth factors in rats.

After intravenous administration of 125I-labeled hepatocyte growth factor (HGF), trichloroacetic acid-precipitable radioactivity in the plasma disappeared rapidly with an early phase half-life of 4 min. The amounts of 125I-HGF distributed to the liver, adrenal, spleen, kidney, and lung tissues were much greater than those that could be accounted for by distribution to the extracellular space alone. The first-pass removal of 125I-HGF by the liver was approximately 26%; the liver accounted for approximately 70% of early-phase removal. The hepatic handling was also analyzed using a single-pass perfused liver system. The steady-state extraction ratio of tracer 125I-HGF was 0.48 but dropped to 0.23 in the presence of excess HGF (135 pM), demonstrating hepatic removal saturation of HGF. In the presence of excess HGF, the heparin-washable 125I-HGF, the heparin-resistant and acid-washable 125I-HGF, and the internalized 125I-HGF dropped to 54, 31, and 32% of the control values. The presence of at least two binding sites for HGF on the liver cell surfaces was made clear: the heparin-washable site and the heparin-resistant and acid-washable binding site, considered to have higher affinity for HGF. The internalization of 125I-HGF was observed to some extent even in the presence of excess HGF and phenylarsine oxide, known to be an inhibitor of polypeptides receptor-mediated endocytosis, suggesting the contribution of a relatively nonspecific internalization mechanism as well as receptor-mediated endocytosis.

Animals↗
PubMed1992

[Endothelial cells and vascular hemostasis].

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.

Cytokines↗
PubMed1992

Effect of benzylpenicillin on the disposition of cefodizime in rats: no net effect on total clearance due to decreased hepatobiliary clearance and increased renal clearance.

Effect of benzylpenicillin (PCG) on the disposition of [14C]cefodizime (CDZM), a nonmetabolizable analog of cefotaxime, was studied in rats. Rats were divided into two groups to receive i.v. injection of [14C]CDZM (15 mg/kg) alone and during the infusion of PCG. Although the total concentration of [14C]CDZM in plasma vs. time data were almost the same between the groups, the unbound concentration of [14C]CDZM was increased 2-fold by the treatment with PCG. Hepatobiliary excretion of [14C]CDZM was reduced by the treatment with PCG, indicating that PCG inhibits the transport of CDZM across the sinusoidal and/or bile canalicular membrane(s). Further support for this hypothesis is the finding that the uptake of [14C]CDZM by the isolated hepatocytes in vitro was via an active transport process that was reduced by PCG. Furthermore, pharmacokinetic analysis of the data obtained from in vivo and in vitro experiments indicated that PCG reduced the permeability of [14C]CDZM across the sinusoidal and bile canalicular membranes to 32% and 40% of the control value, respectively. The urinary excretion of [14C]CDZM was principally via the glomerular filtration process and was not affected by the treatment with PCG. Because of the 2-fold rise in the unbound fraction of [14C]CDZM in the treated rats, the renal clearance for total [14C]CDZM was increased 2-fold by the treatment with PCG. The increase in the renal clearance compensated for the decrease in the hepatic clearance. The net effect of the alterations in these pharmacokinetic parameters resulted in no change in the time profiles of [14C]CDZM concentration in plasma between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Plasmin generation and fibrin(ogen)olysis following desmopressin infusion.

Desmopressin acetate (DDAVP) is known to stimulate the release of tissue-type plasminogen activator (t-PA) from endothelial cells, but it is unclear whether the increased t-PA actually elicits the plasmin generation and fibrin(ogen)olysis in the circulating blood. We measured plasma levels of plasmin-alpha 2-plasmin inhibitor complex, fibrinogen degradation products (FgDP) and fibrin degradation products (FbDP) following desmopressin infusion in 19 patients with bleeding disorders or thrombophilia. Administration of desmopressin (0.3-0.4 microgram/kg) produced a 4.0-fold increase in plasmin-alpha 2-plasmin inhibitor complex at 30 min, whereas neither FgDP nor FbDP was elevated significantly. These findings indicate that desmopressin infusion provokes the generation of plasmin in vivo, but most of the plasmin generated is complexed to alpha 2-plasmin inhibitor and does not degradate fibrin or fibrinogen.

Antifibrinolytic Agents↗
PubMed1991

Circulating thrombomodulin in thrombotic thrombocytopenic purpura.

Endothelial cell injury is thought to be one of the causative factors in thrombotic thrombocytopenic purpura (TTP). A novel index of endothelial injury, plasma thrombomodulin, was measured in 13 patients with acute TTP. The mean plasma concentration of thrombomodulin was elevated in patients with TTP (34.23 +/- 19.08 ng/ml) as compared with healthy subjects (16.99 +/- 2.63 ng/ml, P less than 0.001). Eight (61.5%) of 13 patients had high thrombomodulin values. Markedly elevated thrombomodulin levels were observed in TTP patients who had suffered from systemic lupus erythematosus, in whom plasma thrombomodulin was still elevated when they achieved remission. Five of these 13 patients with TTP had normal plasma levels of thrombomodulin. In addition, the plasma thrombomodulin concentrations were correlated well with von Willebrand factor antigen and tissue-type plasminogen activator antigen levels, both of which are released from stimulated or damaged endothelial cells. No difference was found in plasma thrombomodulin levels between patients who achieved remission and who did not. These findings suggest that the magnitude of the endothelial damage in TTP is variable among patients and that plasma thrombomodulin has limited clinical relevance to the severity of TTP.

Adult↗
PubMed1991

Comparison of the methods for determining cell-surface and intracellular receptors for epidermal growth factor in the rat liver.

We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37 degrees C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intracellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 micrograms/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.

Animals↗
PubMed1991

Kinetic analysis of receptor-mediated endocytosis of epidermal growth factor by isolated rat hepatocytes.

The interaction of epidermal growth factor (EGF) with cell surface receptors and their subsequent endocytosis in isolated rat hepatocytes were analyzed by measuring changes in the concentrations of cell surface-bound, internalized, and degraded EGF. The kinetic model proposed by Wiley and Cunningham (Cell 25: 433-440, 1981) and Gex-Fabry and Delisi [Am. J. Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R768-R779, 1984] was basically utilized for the model analysis. The following kinetic parameters were obtained: association and dissociation rate constants for EGF-receptor interaction, internalization rate constant for EGF-receptor complex (kappa e), internalization rate constant for free receptor (kappa t), sequestration rate constant (kappa s) of the complex from shallow (exchangeable) to deep (nonexchangeable) membraneous compartment, intracellular degradation rate constant and initial cell-surface receptor density. The kappa s value, which was obtained by analyzing the time profiles of EGF association with cells, was approximately 5-10 times larger than the kappa e value determined by directly measuring internalized EGF with the acid-washing technique. This suggests the necessary presence of deep (nonexchanging) compartment of the complex in the plasma membrane. The calculated kappa e value is at least several times larger than the kappa t value, yielding the kinetic basis for the occurrence of receptor downregulation induced by excess EGF. We conclude that, in the overall receptor-mediated processing of EGF after bound to the cell surface receptors, the dissociation process is rapid [half-time (t1/2) less than 1 min], the degradation process is much slower (t1/2 approximately equal to 3 h), and the receptor internalization process is intermediate (t1/2 approximately equal to 6-7 min). In addition, two pools for EGF-receptor complex in the plasma membrane seem to be present, although their identification cannot be made.

Animals↗
PubMed1991

Kinetic analysis of clearance of epidermal growth factor in isolated perfused rat kidney.

Our previous in vivo studies identified the saturable uptake of epidermal growth factor (EGF) by rat kidney (D.C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano, J. Pharm. Sci. 77: 200-207, 1988). In the present study, renal handling of EGF in filtering and nonfiltering isolated perfused rat kidneys was investigated. At designated times after the recirculatory perfusion of 125I-EGF in the nonfiltering kidney, the surface-bound and internalized EGF were separately determined by an acid-washing technique. Time profiles of cell-surface-bound and internalized EGF obtained at the perfusion of a tracer 125I-EGF were fitted to the pharmacokinetic model, and kinetic parameters obtained were as follows: konRs = 0.49 ml.min-1.g-1, koff = 0.87 min-1, kint = 0.20 min-1 where konRs is the binding clearance of EGF with its receptor and koff and kint represent the dissociation and internalization rate constants of the EGF-receptor complex, respectively. The Scatchard analysis of the concentration-dependent EGF binding in the nonfiltering kidney suggests the presence of two binding components, one with high affinity [the apparent dissociation constant (Kd1 = 0.1 nM) and the other with low affinity (Kd2 = 30 nM]. By comparing the internalization clearance (CLi) with filtering and nonfiltering kidneys, we concluded that the renal uptake of EGF occurs mainly from the antiluminal side via receptor-mediated endocytosis in a saturable manner and that the nonsaturable reabsorption of EGF from the luminal membrane after glomerular filtration is relatively small. The contribution of reabsorption, however, becomes larger with the increase in EGF concentration.

Absorption↗
PubMed1991

Kinetic evaluation for measurement of in vivo receptor occupancy by psychotropic drug in brain: implication for human studies.

In vivo receptor occupancy of psychotropic drugs in brain can be estimated by measuring the tissue radioactivity of the tracer, which binds specifically to the receptor unoccupied by the drugs (back titration method). In this study, the validity of this method was evaluated by computer simulation, using various values for the plasma elimination rate, rate of transport across the blood-brain barrier, rate of receptor association and dissociation for both drug and tracer, and the sampling time. The differential equations based on a nonlinear three-compartment model including a plasma pool, precursor pool, and specific binding pool were solved numerically by the Runge-Kutta-Gill method. The receptor occupancy calculated by this method was close to the true value when the plasma concentration and specific binding fraction of the drug did not change greatly during circulation of the tracer. Although the error in calculated occupancy at 5 min after the tracer administration was smaller than that at 20 min, tracer may not greatly accumulate in brain tissue during the initial 5 min in some situations. Our analysis shows that it is necessary to adequately control the elimination rate of drug from plasma and to allow sufficient time for radioactivity to accumulate in the tissue. Therefore, this method requires previous knowledge of the pharmacokinetic behavior of both the drug and the tracer in plasma and tissue. The operation scheme that we suggest for the accurate measurement of the receptor occupancy in vivo can be used in human studies with positron emission tomography and may be useful for therapeutic drug monitoring.

Brain↗
PubMed1991

Kinetic evaluation of pharmacological effects based on allosteric coupling of the benzodiazepine/gamma-aminobutyric acidA receptor in the brain.

A mathematical allosteric coupling model has been proposed to describe the process by which binding to the benzodiazepine/gamma-aminobutyric acid (GABA) receptor complex initiates a biological response. The model states that the first receptors (benzodiazepine receptors) can diffuse independently in the plane of the membrane and reversibly associate with the second receptors (GABA receptors) to regulate their activity (induction of increased chloride ion flux due to the opening of the chloride ion channel). The ratio of agonist-bound to total GABA receptor density was defined to be directly proportional to the biological response in the model. The analysis makes the following assumptions: i) the binding affinity of agonists (muscimol or benzodiazepine) to the benzodiazepine receptor/GABA receptor complex is much greater than that to each receptor alone; ii) the double receptor-single agonist (benzodiazepine, muscimol or GABA) ternary complex binds to the other agonist with a high binding affinity as compared with that of each agonist to an agonist-free receptor complex; iii) benzodiazepine receptor-GABA receptor interaction is enhanced in the presence of each agonist; iv) the GABA receptor is desensitized after the binding of GABA agonist (GABA or muscimol) to the receptor. The modeling exercise shows that the benzodiazepine concentration required for half-maximal biological response is lower than that required for half-maximal receptor binding. In the case of the GABA agonist, a linear relationship between receptor occupancy and biological response was observed. The degree of discrepancy between the two profiles (receptor occupancy and biological response) concerning benzodiazepine concentration dependency and time dependency increased with a decrease in the dissociation constants based on the benzodiazepine receptor-GABA receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗
PubMed1991

[Evaluation of clinical usefulness of a rapid quantitative measurement of D dimer (cross-linked fibrin degradation products)].

In order to evaluate precisely the fibrinolytic states in clinical disorders, plasma levels of D dimer (cross-linked fibrin degradation products) were measured by a newly developed, rapid quantitative method based on the latex photometric immunoassay in patients with hematological malignancies, diabetes mellitus, collagen disease, liver disease, thrombotic disease and disseminated intravascular coagulation (DIC). Plasma levels of D dimer were elevated in a variety of diseases, especially in DIC. Patients with hematological malignancies, liver disease and thrombotic disease also had relatively high levels of D dimer. On the whole, D dimer values were positively correlated with plasmin-alpha 2-plasmin inhibitor complex and thrombin-antithrombin III complex. In addition, plasma D dimer was measured during fibrinolytic therapy with urokinase or tissue-type plasminogen activator; its elevation was detected in some patients. These findings indicate that accelerated fibrinolysis is frequently observed in a variety of diseases, and that a rapid quantitative measurement of D dimer would be valuable for the precise assessment of fibrinolysis in these disease states.

Disseminated Intravascular Coagulation↗