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Background: Self-guided web-based interventions have the potential of addressing help-seeking barriers and symptoms common among university students, such as depression and anxiety. Unfortunately, self-guided interventions are also associated with less adherence, implicating motivation as a potential moderator for adherence and improvement for such interventions. Previous studies examining motivation as a moderator or predictor of improvement on web-based interventions have defined and measured motivation variably, producing conflicting results., Objective: This secondary analysis of data from a randomized controlled trial aimed to examine constructs of motivation as moderators of improvement for a self-guided 8-week web-based intervention in university students (N=1607)., Methods: Tested moderators included internal motivation, external motivation, and confidence in treatment derived from the Treatment Motivation Questionnaire. The primary outcome was an improvement in depression and anxiety measured by the Depression Anxiety Stress Scale-21., Results: Piecewise linear mixed effects models showed that internal motivation significantly moderated symptom change for the intervention group (t 1504 =-2.94; P=.003) at average and high (+1 SD) motivation levels (t 1507 =-2.28; P=.02 and t 1507 =-4.05; P<.001, respectively). Significant results remained even after controlling for baseline severity. The results showed that confidence in treatment did not significantly moderate symptom change for the intervention group (t 1504 =1.44; P=.15). In this sample, only internal motivation was positively correlated with service initiation, intervention adherence, and intervention satisfaction., Conclusions: The combination of a web-based intervention and high or moderate internal motivation resulted in greater improvement in the total Depression Anxiety Stress Scale-21 score. These findings highlight the importance of conceptually differentiating motivation-related constructs when examining moderators of improvement. The results suggest that the combination of a web-based intervention and high or moderate internal motivation results in greater improvement. These findings highlight the importance of conceptually differentiating motivation-related constructs when examining moderators of improvement. To better understand the moderating role of internal motivation, future research is encouraged to replicate these findings in diverse samples as well as to examine related constructs such as baseline severity and adherence. Understanding these characteristics informs treatment strategies to maximize adherence and improvement when developing web-based interventions as well as allows services to be targeted to individuals likely to benefit from such interventions., Trial Registration: ClinicalTrials.gov NCT04361045; https://clinicaltrials.gov/study/NCT04361045., (©Maria Hanano, Leslie Rith-Najarian, Elizabeth Gong-Guy, Denise Chavira. Originally published in JMIR Formative Research (https://formative.jmir.org), 03.07.2024.)

Incisional scarring is a factor of cosmetic appearance evaluated after breast reconstruction, along with the shape, position, and size of the breast. This study aimed to examine the effect of the incision scar location on patient satisfaction after breast reconstruction. Using the Japanese version of the SCAR-Q, we assessed the scar appearance, symptoms and psychosocial effects. Plastic surgeons performed assessments using the Manchester Scar Scale. The patients were divided into two groups: those with scars on the margins of the breast (MB group) and those with scars in the breast area (IB group). The results revealed that patients in the MB group reported significantly higher satisfaction with the scar appearance and psychological impact than those in the IB group. However, assessments using the Manchester Scar Scale did not reveal any significant differences between the two groups. In conclusion, this study underscores the importance of patient-reported outcomes in the evaluation of scar satisfaction after breast reconstruction. Patients tend to prefer and have higher satisfaction with scars along the breast margin, which offers valuable insights into surgical decisions. Further studies with larger and more diverse sample sizes are required for validation., (© 2024 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Introduction: Opioid overdose deaths are increasing rapidly in the United States. Medications for opioid use disorder (MOUD) are effective and can be delivered in primary care, but uptake has been limited in rural communities. Referral to and coordination with an external telemedicine (TM) vendor by rural primary care clinics for MOUD (TM-MOUD) may increase MOUD access for rural patients, but we know little about perspectives on this model among key stakeholders. As part of a TM-MOUD feasibility study, we explored TM-MOUD acceptability and feasibility among personnel and patients from seven rural primary care clinics and a TM-MOUD vendor., Methods: We conducted virtual interviews or focus groups with clinic administrators (n = 7 interviews), clinic primary care and behavioral health providers (8 groups, n = 30), other clinic staff (9 groups, n = 37), patients receiving MOUD (n = 16 interviews), TM-MOUD vendor staff (n = 4 interviews), and vendor-affiliated behavioral health and prescribing providers (n = 17 interviews). We asked about experiences with and acceptability of MOUD (primarily buprenorphine) and telemedicine (TM) and a TM-MOUD referral and coordination model. We conducted content analysis to identify themes and participants quantitatively rated acceptability of TM-MOUD elements on a 4-item scale., Results: Perceived benefits of vendor-based TM-MOUD included reduced logistical barriers, more privacy and less stigma, and access to services not available locally (e.g., counseling, pain management). Barriers included lack of internet or poor connectivity in patients' homes, limited communication and trust between TM-MOUD and clinic providers, and questions about the value to the clinic of TM-MOUD referral to external vendor. Acceptability ratings for TM-MOUD were generally high; they were lowest among frontline staff., Conclusions: Rural primary care clinic personnel, TM-MOUD vendor personnel, and patients generally perceived referral from primary care to a TM-MOUD vendor to hold potential for increasing access to MOUD in rural communities. Increasing TM-MOUD uptake requires buy-in and understanding among staff of the TM-MOUD workflow, TM services offered, requirements for patients, advantages over clinic-based or TM services from clinic providers, and identification of appropriate patients. Poverty, along with patient hesitation to initiate treatment, creates substantial barriers to MOUD treatment generally; insufficient internet availability creates a substantial barrier to TM-MOUD., Competing Interests: Declaration of competing interest None., (Copyright © 2023 RAND Corporation. Published by Elsevier Inc. All rights reserved.)

The risks of concomitant benzodiazepine (BZ) and opioid use are significant. Despite the urgent need to reduce BZ use among patients taking opioids, no treatment intervention research to our knowledge has addressed treatment for this concurrent, high-risk use. The current study will evaluate the efficacy of augmenting BZ taper procedures with CBT for anxiety disorders that has been adapted specifically for patients with concomitant BZ and opioid use (either use as prescribed or misuse), a high-risk patient population. Research combining rapidly scalable behavioral interventions ancillary to pharmacological approaches delivered via telehealth in primary care settings is innovative and important given concerning trends in rising prevalence of BZ/opioid co-prescription, BZ-associated overdose deaths, and known barriers to implementation of behavioral health interventions in primary care. CBT delivery using telehealth has the potential to aid adherence and promote access and dissemination of procedures in primary care. Lastly, the current study will utilize an experimental therapeutics approach to preliminarily explore the mechanism of action for the proposed interventions. The overall aim of the present pilot randomized controlled trial is to examine the feasibility and preliminary efficacy of a BZ taper with CBT for anxiety disorders adapted for patients with concomitant BZ (BZT + CBT) and opioid use to a BZ taper with a control health education program (BZT + HE) in a sample of individuals (N = 54) who have been prescribed and are taking benzodiazepines and opioids for at least 3 months prior to baseline and experience anxious distress. Screening and outcome measures, methods, and implications are described. Trial Registration: ClinicalTrials.gov (NCT05573906)., Competing Interests: Declaration of Competing Interest Dr. Otto is compensated for his role on the scientific and clinical advisory boards for Big Health. Dr. Mooney receives research support from Aelis Pharma. There are no other declarations of interest to report., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: The use of telemedicine (TM) has accelerated in recent years, yet research on the implementation and effectiveness of TM-delivered medication treatment for opioid use disorder (MOUD) has been limited. This study investigated the feasibility of implementing a care coordination model involving MOUD delivered via an external TM provider for the purpose of expanding access to MOUD for patients in rural settings., Methods: The study tested a care coordination model in 6 rural primary care sites by establishing referral and coordination between the clinic and a TM company for MOUD. The intervention spanned approximately 6 months from July/August 2020 to January 2021, coinciding with the peak of the COVID-19 pandemic. Each clinic tracked patients with OUD in a registry during the intervention period. A pre-/post-intervention design (N = 6) was used to assess the clinic-level outcome as patient-days on MOUD based on patient electronic health records., Findings: All clinics implemented critical components of the intervention, with an overall TM referral rate of 11.7% among patients in the registry. Five of the 6 sites showed an increase in patient-days on MOUD during the intervention period compared to the 6-month period before the intervention (mean increase per 1,000 patients: 132 days, P = .08, Cohen's d = 0.55). The largest increases occurred in clinics that lacked MOUD capacity or had a greater number of patients initiating MOUD during the intervention period., Conclusions: To expand access to MOUD in rural settings, the care coordination model is most effective when implemented in clinics that have negligible or limited MOUD capacity., (© 2023 The Authors. The Journal of Rural Health published by Wiley Periodicals LLC on behalf of National Rural Health Association.)

Background: Self-guided online interventions offer users the ability to participate in an intervention at their own pace and address some traditional service barriers (eg, attending in-person appointments, cost). However, these interventions suffer from high dropout rates, and current literature provides little guidance for defining and measuring online intervention adherence as it relates to clinical outcomes., Objective: This study aims to develop and test multiple measures of adherence to a specific self-guided online intervention, as guided by best practices from the literature., Methods: We conducted secondary analyses on data from a randomized controlled trial of an 8-week online cognitive behavioral program that targets depression and anxiety in college students. We defined multiple behavioral and attitudinal adherence measures at varying levels of effort (ie, low, moderate, and high). Linear regressions were run with adherence terms predicting improvement in the primary outcome measure, the 21-item Depression, Anxiety, and Stress Scale (DASS-21)., Results: Of the 947 participants, 747 initiated any activity and 449 provided posttest data. Results from the intent-to-treat sample indicated that high level of effort for behavioral adherence significantly predicted symptom change (F4,746=17.18, P<.001; and β=-.26, P=.04). Moderate level of effort for attitudinal adherence also significantly predicted symptom change (F4,746=17.25, P<.001; and β=-.36, P=.03). Results differed in the initiators-only sample, such that none of the adherence measures significantly predicted symptom change (P=.09-.27)., Conclusions: Our findings highlight the differential results of dose-response models testing adherence measures in predicting clinical outcomes. We summarize recommendations that might provide helpful guidance to future researchers and intervention developers aiming to investigate online intervention adherence., Trial Registration: ClinicalTrials.gov NCT04361045; https://clinicaltrials.gov/ct2/show/NCT04361045., (©Maria Hanano, Leslie Rith-Najarian, Meredith Boyd, Denise Chavira. Originally published in JMIR Mental Health (https://mental.jmir.org), 28.03.2022.)

The involvement of P-glycoprotein (P-gp) in pentazocine (PTZ) transport at the blood-brain barrier (BBB) in rats was evaluated by means of an in vivo study using the brain uptake index (BUI) method. The amount of radioactivity in the brain was estimated at different intervals (up to 240 s) after carotid injection in rats. The apparent elimination rate constant (k(test)) due to efflux of PTZ from the brain was calculated as 0.22 min(-1). The observed BUI values of [(3)H]-PTZ (0.35 microM) were not significantly different between 5 and 15 s after the carotid injection. The concentration-dependent uptake of PTZ by the brain was increased gradually by increasing the concentration (0.01-1 mM) of PTZ in the injection solution. The apparent uptake of PTZ by the brain increased in the presence of P-gp inhibitors such as cyclosporin A, quinidine, verapamil and vinblastine after the carotid injection. These results suggest that the increment of PTZ uptake by the brain could be explained by the saturable efflux transport system involving a P-gp-mediated efflux mechanism of PTZ transport at the BBB.

To characterize pentazocine (PTZ) transport across the blood-brain barrier (BBB), the cerebrovascular permeability-surface area product (PS(inf)) of PTZ was determined by a well-established in situ rat brain perfusion technique. The uptake kinetics of PTZ by the rat brain exhibited saturability, which indicates the simultaneous mechanisms of carrier-mediated transport and passive diffusion. The kinetic parameters were estimated as follows: maximal influx rate (V(max)), 27.2 +/- 5.2 nmol/s/g brain; apparent Michaelis constant (K(m)) for the saturable component of PTZ uptake, 2.9 +/- 0.5 mM; nonsaturable uptake rate constant (K(d)), 1.5 +/- 0.3 microL/s/g brain. BBB transport of PTZ was significantly inhibited by cationic drugs such as diphenhydramine, propranolol, and eptazocine (a narcotic-antagonist analgesic), but not by choline, suggesting that the PTZ transport system is shared by cationic drugs. Furthermore, co-perfusion of verapamil caused a significant (two-fold) increase in the BBB permeability to PTZ. This finding indicates that PTZ may be a substrate of the endogenous BBB efflux transport system, P-glycoprotein. These findings demonstrate that the primary mechanism governing the uptake of PTZ by the brain is carrier-mediated transport, not passive diffusion., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2346-2353, 2002)

The influx transport mechanism of pentazocine (PTZ) at the blood-brain barrier (BBB) was investigated in rats using the carotid injection technique. The uptake kinetics of PTZ into the rat brain exhibited saturability, which occurred by both nonsaturable and carrier-mediated transport processes. The in vivo kinetic parameters were estimated as follows: the maximal uptake rate (Jmax), 3.6 +/- 1.2 micromol/min/g brain and the apparent Michaelis constant (K1), 3.7 +/- 1.7 mM for the saturable component of PTZ into the brain, and the nonsaturable uptake rate constant (Kd), 0.06 +/- 0.04 ml/min/g brain. The uptake of PTZ by the brain was strongly inhibited by lidocaine, imipramine and propranolol, and also by H1-antagonists such as mepyramine, diphenhydramine. In addition, narcotic-antagonist analgesic (buprenorphine, butorphanol or eptazocine) and an opioid antagonist (naloxone) significantly inhibited PTZ transport. These results suggest that PTZ permeates into the brain via a carrier-mediated transport system, which may widely recognize the cationic drugs.

Nifedipine-containing calcium phosphate cement (CPC) was prepared, and nifedipine (NF) release from this preparation was evaluated by the shaking method (SK), Japanese Pharmacopoeia XIV (JPXIV) paddle method (PD), and JPXIV flow-through cell method (FT). The release of NF from the CPC preparation continued for 7 d or longer by all these methods. This suggests that the release of NF can be controlled by preparing NF-containing CPC. The release pattern of NF from CPC in these tests was found to follow the Higuchi equation. However, the Higuchi constant differed among the three dissolution tests, probably because the apparent tortuosity of capillary system (tau) varied.

The relationship between the pharmacokinetics and the pharmacodynamics of eptazocine, a narcotic-antagonist analgesic, was investigated in rats. The analgesic effect of eptazocine (2.5, 5 and 10 mg/kg) following intravenous (i.v) administration was evaluated by both the Randall-Selitto method and the D'Amour-Smith method. The analgesic effects were determined before and at designed intervals for a period of 120 min after eptazocine administration, and are expressed as area under the effect-time curve (AUC(E)). The plasma concentration of eptazocine was determined by fluorescence HPLC and was analyzed with a two compartment open model using the nonlinear least-squares method. Eptazocine produced a dose-dependent analgesic effect. It was demonstrated that eptazocine has a linear relationship between AUC(E) and the area under the plasma concentration-time curve (AUC) following i.v. administration for three different doses ranging from 2.5 to 10 mg/kg.

The effect of naloxone, a potent and specific opioid antagonist, on cerebral glucose utilization was investigated in conscious rat. For quantitative evaluation of the functional activity in brain, the regional cerebral metabolic rate for glucose (rCMRglc) was measured by the double tracer technique, using [14C]2-deoxyglucose and [3H]3-o-methylglucose. Intravenous administration of naloxone significantly increased rCMRglc in the medulla and thalamus at a dose of 1 or 10 mg/kg, and in the cerebral cortex, midbrain and cerebellum at a dose of 10 mg/kg. Our findings strongly suggest that naloxone by itself affects the cerebral functional activity within 10 min of administration.

Transport characteristics of 17beta-estradiol 17beta-D-glucuronide (E217betaG), a dual substrate of the transporters for cellular uptake (organic anion-transporting polypeptide 1 or oatp1) and cellular excretion (multidrug resistance-associated protein 1or MRP1), in the rat choroid plexus were studied in vivo and in vitro. The uptake of E217betaG into isolated choroid plexus was mediated by an energy-dependent system with a Km of 3.4 microM. Together with the previous finding that oatp1 is localized on the apical membrane of choroid plexus, these results suggest that oatp1 is responsible for the uptake of this ligand. After intracerebroventricular administration, elimination of E217betaG from cerebrospinal fluid was probenecid sensitive and much more rapid than that of inulin; less than 2% of the administered E217betaG and 40 to 50% of inulin remained in the cerebrospinal fluid 20 min after intracerebroventricular administration. In addition, the amount of E217betaG associated with choroid plexus at 20 min was negligible, suggesting the presence of an efficient excretion system on the basolateral membrane of choroid plexus. Expression of MRP1 was detected in choroid plexus. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analyses indicated that the expression level of MRP1 in choroid plexus is about four or five times higher than that in the lung, one of the tissues exhibiting high expression of MRP1. Together with the in vivo vectorial transport of E217betaG, these results can be accounted for by assuming that there is basolateral localization of MRP1 in choroid plexus. Combined, oatp1 and MRP1 may synergistically mediate the efficient transcellular transport of E217betaG across choroid plexus.

This study aimed to investigate the behavior of an endogenous beta-endorphin (beta-EP) in the brain after subcutaneous (s.c.) injection of carrageenin or intravenous (i.v.) injection of indomethacin (IDM). The carrageenin was injected into rat hind paw subcutaneously in order to evoke only a local nociceptive stimulus. The beta-EP concentration in the brain region was determined by radioimmunoassay at designated sampling times after the injection. It was observed that the beta-EP concentration in the midbrain declined from 2.8+/-0.3 at 1 h to 1.3+/-0.02 ng/mg protein at 9 h. After the s.c. injection of carrageenin, the beta-EP concentrations in the midbrain were found to be closely related to the nociceptive sensitivity which was determined by the Randall-Selitto test. On the other hand, a significant elevation of the beta-EP concentration was observed in the hypothalamus from 3 h until 5 h compared with that of control. IDM was injected into rats at doses of 2.9, 5.8 and 8.6 mg/kg via the femoral vein. After i.v. administration of IDM, the beta-EP increased in the hypothalamus, medulla oblongata, and midbrain, depending on the doses used. The value of hypothalamic beta-EP concentration was two times higher than that of carrageenin. We found that nociceptive stimuli and IDM brought a change in the beta-EP concentration in the brain of rats.

In order to assess quantitatively the pharmacodynamic process of pentazocine (PTZ), time courses of its plasma concentration and of the occupation of specific opioid receptors in the brain were investigated after intravenous (i.v.) administration of PTZ to rats. The plasma concentration of PTZ was determined by HPLC and the pharmacokinetic parameters were analyzed using nonlinear least-squares analysis. Measurement of ex vivo receptor occupation was made by comparing the specific [3H]naloxone (opioid receptor antagonist) binding in vitro to the crude P2-synaptosomal fractions between vehicle-treated rats (control) and PTZ-treated rats. Following the i.v. administration of PTZ, the occupancy of specific opioid receptors decreased rapidly until 10 min, depending on the two pharmacological doses (2.5 and 10 mg/kg). The results strongly suggest the fast binding kinetics of PTZ in terms of its association with and dissociation from specific opioid receptor sites in the brain in addition to its fast rate of disappearance from the brain compartment. Furthermore, we demonstrated that the time profile of receptor occupancy correlated well (r = 0.8650) with that of the unbound concentration in plasma until 120 min after the i.v. administration of PTZ to rats.

The effects of buprenorphine (BNP, 10-200 micrograms/kg, i.v.) and pentazocine (PTZ, 2.5-10 mg/kg, i.v.) on the regional cerebral metabolic rate for glucose (rCMRglc) were analyzed in nine anatomically discrete areas of the conscious rat brain by the simultaneous use of [14C]2-deoxyglucose, the glucose analogue that can be phosphorylated in the brain, and [3H]3-O-methylglucose, a nonmetabolizable glucose analogue. Originally, this method was developed by Gjedde and Diemer in the rat and in humans. The rCMRglc was significantly decreased by BNP (100 or 200 micrograms/kg) in most of the brain regions investigated, except the cerebellum. In contrast, PTZ (10 mg/kg) significantly increased rCMRglc in the cerebral cortex and medulla. In the cerebral cortex and medulla, the direction of the effect on rCMRglc was opposite for BNP (22% decrease at the dose of 200 micrograms/kg) and PTZ (22% increase at the dose of 10 mg/kg). These findings strongly suggest that the discrepancies between the marked effects of BNP (a partial mu agonist and kappa antagonist) and PTZ (a mu antagonist and kappa agonist) on rCMRglc reflect the selectivity of agonist action at the different types of opioid receptors, mu and kappa receptors, in the rat brain.

Purpose: The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells., Methods: The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface. 125I-EGF was prebound to the basal surface at 4 degrees C, followed by extensive washing and subsequent incubation at 37 degrees C in EGF-free medium., Results: Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (approximately 90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1-3 min, followed by intracellular degradation with a half-life of 20-50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130-250 min. Even when 125I-EGF was incubated with MDCK cells at 37 degrees C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160-170 min., Conclusions: The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.

The relationship between the pharmacokinetic properties and the analgesic effect of indomethacin (IDM) was evaluated on a carrageenin-induced inflammation model in the rat. Rats were administered the drug in one of two ways: intravenous (i.v.) IDM bolus or i.v. IDM infusion. The analgesic activity was measured by Randall-Selitto test. No correlation was observed between the analgesic effect and the plasma IDM concentration after i.v. bolus administration of IDM. However, in the case of infusion, IDM produced a dose-dependent analgesic effect. In this paper, we demonstrated that the plasma concentration of IDM maintained by i.v. infusion had a prolonged analgesic effect on the carrageenin-induced inflammation model.

To clarify the contribution of P-glycoprotein to the biliary excretion of vincristine in rats, the effects of induction of hepatic P-glycoprotein by a phenothiazine treatment on the biliary excretion of [3H]vincristine were investigated. Immunoblot analysis using C219, a monoclonal antibody to P-glycoprotein, demonstrated that the phenothiazine treatment increased the P-glycoprotein level in isolated bile canalicular membrane vesicles approximately 6.5-fold. Transport of [3H]vincristine to canalicular membrane vesicles from the phenothiazine-treated and control rats revealed ATP-dependency, with an overshoot that results from the consumption of medium ATP. The maximum ATP-dependent uptake was increased in canalicular membrane vesicles from the phenothiazine-treated rats approximately 2-fold compared to the control. The biliary excretion of [3H]vincristine was further studied using an indicator dilution method in a single-pass perfused liver. The ratios of the cumulative amount of [3H]vincristine excreted into the bile ot the amount of [3H]vincristine taken up by the liver at 15, 30 and 90 min were significantly increased in the phenothiazine-treated rats by 60, 45 and 25%, respectively, compared to the control rats. Furthermore, the corrected mean residence time of [3H]vincristine in hepatocytes in the phenothiazine-treated rats was reduced to 21 min from that in the control rats (30 min), supporting the contention that the induction of hepatic P-glycoprotein on the bile canalicular membrane function sas a transporter not only in the isolated membrane but also in the more physiological perfused liver system. One must be cautious in the interpretation of the data, however, since phenothiazine can induce other proteins which might affect the behavior of [3H]vincristine.

Plasma levels of urokinase-type plasminogen activator (u-PA) were measured with an enzyme-linked immunosorbent assay in patients with leukemias. As compared with healthy subjects (0.73 +/- SD 0.17 ng/ml), plasma u-PA antigen level was markedly elevated in patients with acute promyelocytic leukemia (APL) (1.76 +/- 0.89 ng/ml) at disease onset. Mean u-PA concentrations in patients with other acute nonlymphoblastic leukemia (0.57 +/- 0.51 ng/ml), acute lymphoblastic leukemia (0.77 +/- 0.82 ng/ml) and chronic myelocytic leukemia in blastic crisis (1.30 +/- 1.35 ng/ml) were not significantly elevated, but some of them showed an elevation of plasma u-PA. Plasma u-PA values were correlated with some of the fibrinolytic parameters such as FDP and D-dimer. Plasma u-PA antigen was decreased after the administration of antileukemic drugs in patients with APL. These results suggest that the coagulopathy in patients with various leukemias may in part be associated with u-PA release from the leukemic cells, especially in patients with APL.

The influx clearance (PSinf.MID) of the weak acid 5,5'-dimethyl-oxazolidine-2,4-dione (DMO) was determined by the multiple indicator dilution method with the isolated perfused rat liver under various perfusate pH conditions, ranging from 6.4 to 7.6. Although the pH partition theory predicted an increase in influx clearance of ten times in proportion to the change in the unionized fraction of DMO, there was no measurable change in this value. The effect of medium pH on the steady-state cell/medium concentration ratio (C/M) ratio of DMO was also investigated using isolated hepatocytes. The C/M ratio increased while medium pH decreased, but this change was less marked than predicted by the pH partition theory. Finally the pH dependency of the dissociation rate constant (koff) of epidermal growth factor from its receptor was also investigated using both isolated rat hepatocytes and the perfused rat liver. When the extracellular pH was changed from 6.4 to 5.6, the koff value of isolated hepatocytes increased 44 times, while that of the perfused rat liver increased only 9 times. Therefore, the effect of changing the extracellular pH on pH-dependent dissociation of epidermal growth factor from its cell-surface receptor was less in the perfused liver than in isolated hepatocytes. These findings, in addition to the well-known existence of the Na(+)-H+ exchanger on the sinusoidal membrane and the possible existence of the unstirred water layer in the Disse space, seem to suggest the existence of the proton diffusion barrier in the rat liver, which remains stronger in the perfused liver than in isolated hepatocytes.

We present a 57-year-old man with end-stage renal failure due to chronic glomerulonephritis, who had been on hemodialysis for 13.5 years and had suffered from recurrent painful swelling of the left leg for 4.7 years. A diagnosis of deep venous thrombosis was made by the phlebography. Coagulation studies showed decreased protein C activity despite a normal protein C antigen level. None of his relatives had decreased protein C activity, and the levels of the other coagulation factors synthesized by the liver were all normal. Accordingly, the patient was diagnosed as having acquired type II protein C deficiency.

The effect of muscimol, a GABAA receptor agonist, on the basic metabolic activity was investigated in the mouse brain and was correlated with its receptor occupancy. For the quantitative evaluation of the functional activity of the mouse brain, the cerebral glucose utilization was measured by the double tracer technique, using [14C] 2-deoxyglucose and [3H]3-O-methylglucose. The dose-dependent reduction in the cerebral glucose utilization was observed after intravenous administration of various doses of muscimol (0.3-1.5 mg/kg). On the other hand, the GABAA receptor occupancy of muscimol was determined by using the values of the unbound drug concentration in the brain tissue and the receptor dissociation constant based on the in vitro binding experiments using the dissociated brain cells. The tissue unbound concentration of muscimol was calculated by multiplying the total concentration in the brain after administration of muscimol and the tissue unbound fraction, which was measured by the equilibrium dialysis method using brain tissue homogenate. A linear relationship was observed between the GABAA receptor occupancy of muscimol and the decrease in the cerebral glucose utilization. This finding indicates that the simple receptor occupancy theory holds for this receptor-ligand system, and there is a large difference in the effect on glucose metabolic response between GABAA receptor-agonist interaction and benzodiazepine receptor-agonist interaction.

A novel cell line (KH88) was established from a patient with chronic myelogenous leukemia in blastic crisis. The leukemic blasts had the features of undifferentiated blasts with basophilic agranular cytoplasm and they were focally positive for acid phosphatase and alpha-naphthyl acetate esterase. CD36, CD33, HLADR, and CD71 were expressed on the surfaces of the blast cells. Most blasts were positive for platelet peroxidase activity, and some of them had granules containing aggregates of ferritin molecules. These findings were compatible with those of 'early' erythroblastic leukemia, this established cell line (KH88) having similar characteristics, and actually producing hemoglobin A and hemoglobin F. Although the KH88 cells were negative for megakaryocytic markers, they were induced to express CD41 by phorbol ester. Further, a few KH88 cells were positive for myeloperoxidase. This cell line was thus revealed to have the capacity to differentiate into three lineages, providing a useful model for studying the differentiation of multipotential stem cells. Moreover, a subline of KH88 had a peculiar chromosome abnormality, del(3)(q21q25); it would be useful to study the significance of this chromosomal abnormality.

Concentration-dependency in the hepatic elimination of l-propranolol (l-PR) was investigated over a wide range of concentrations from 60 to 2200 microM in an isolated rat liver perfusion system. Under the steady-state condition produced by unlabeled l-PR at various concentrations, 3H-l-PR and 14C-inulin were bolusly injected into the portal vein, and the outflow was collected at 0.5 s intervals over 30 s. Up to 300 microM, the instantaneous hepatic availability of l-PR was approximately 4%, while it abruptly increased when the perfusate concentration exceeded 300 microM. To determine which process (influx or efflux or sequestration process) caused the nonlinearity, we calculated the rate constants k1 (influx), k2 (efflux), and k3 (sequestration) based on the "distributed" model. With increasing l-PR concentration in the perfusate, k2 increased approximately two times, whereas k3 decreased to approximately one-half. In contrast, k1 was independent of the perfusate concentration. The concentration-dependency of k2 was explained by saturation of l-PR tissue binding, since the tissue unbound fraction of l-PR obtained with liver homogenates and isolated hepatocytes increased approximately two times. The efflux and sequestration clearances were then normalized by the unbound fractions in the liver. The efflux clearance for unbound l-PR was constant irrespective of the perfusate concentration, whereas the sequestration clearance for unbound l-PR (CL(int) showed Michaelis-Menten type saturation (Km = 28 microM, Vmax = 2.8 mumol/min/g liver, alpha (nonspecific) = 2- ml/min/g liver).(ABSTRACT TRUNCATED AT 250 WORDS)

We kinetically examined tissues responsible for the conjugative metabolism (glucuronidation and sulfation) of a component in a crude drug, liquiritigenin (LG; 2,3-dihydro-7-hydroxy-2-(4- hydroxyphenyl)-(S)-4H-1-benzopyran-4-one) in rats in vivo. LG has been found to form five kinds of conjugates (4'-O-glucuronide (M1), 7-O-glucuronide (M2), 4',7-O-disulfate (M3), 4'-O-glucuronide-7-O-sulfate (M4) and 7-O-glucuronide-4'-O-sulfate (M5)). Analysis based on metabolite kinetics [K. S. Pang, J. Pharmacokin. Biopharm., 13, 633 (1985)] of the area under the plasma concentration curves (AUCplasma) and cumulative biliary excretions (Aibile) of the ligands after intravenous or hepatic portal venous administration of LG revealed that the liver has the ability to generate all the metabolites. For M1 and M2, the apparent biliary excretion clearance (CLbile,app) obtained by dividing the biliary excretion rate for the metabolite by the plasma concentration of the metabolite decreased with time, confirming that M1 and M2 were formed in the liver. To further analyze the formation rate constants for metabolites in each tissue, we measured the ligand content in several tissues after intravenous administration of LG. By correcting the content of metabolites that were taken up from the plasma, we found that the formation rates per gram of tissue were largest in the liver, except for M3. The metabolic capability of the kidney for M1 and M2 was 15% and 60%, respectively, to that of the liver whereas for M3, the metabolic ability of the kidney was 2.5-fold greater than that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon has been demonstrated to stimulate the uptake of bile acid in isolated rat hepatocytes (Am. J. Physiol., 249, G427 (1985)). In the present study, we determined the influence of glucagon on the hepatic transport of a bile acid, taurocholate (TCA), in isolated rat livers. A single-pass perfusion and a rapid-injection, multiple indicator dilution method were employed. The hepatic availability at steady-state was 0.04. With the presence of glucagon in the perfusate (from 10(-9) to 10(-7) M), the bile flow rate was stimulated by 30%, while hepatic availability was decreased from 0.04 to 0.02 with a stepwise increase in glucagon concentration. Thirty min after the infusion of glucagon (300 nM), [3H]TCA and [14C]inulin were injected in a bolus state into the portal vein, and the outflow was collected at 1.0 s intervals over 30 s. Glucagon decreased the instantaneous hepatic availability by 50% compared to the control level, and was thus compatible with the steady-state experiments. In the control experiment, the influx clearance (PSinf) was 20 times higher than the efflux clearance (PSeff). Glucagon (300 nM) in the perfusate enhanced PSinf by 50% of the control, whereas sequestration clearance (CLseq) and the biliary excretion rate constant remained unchanged. PSeff was stimulated to 2 times the control, but still remained much smaller than CLseq. Based on the comparison of PSinf, PSeff and CLseq, the rate-determining process of TCA hepatic elimination was the influx process in both the presence and absence of glucagon. Taken together, the enhancement of the influx process was responsible for the decrease in TCA hepatic availability caused by glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)

Rilmazafone (RZ) is an orally active sleep inducer which can be activated to its cyclic form (M1) via the labile desglycylated metabolite (DG). In this scheme, RZ is exclusively metabolized to DG and M1 by aminopeptidases in the small intestine. The concentration of M1 in the systemic plasma after oral administration of RZ has been reported to be higher than that observed after administration of M1, due to the lower hepatic extraction of DG than M1 (Koike et al., Drug Metab. Dispos., 16, 609 (1988)). In the present study, the disposition of DG and M1 in rat liver was investigated, using the multiple indicator dilution method. The hepatic availabilities (F) of DG and M1, assessed from the recovery into the hepatic vein, were 0.16 and 0.07, respectively, which was consistent with the previous in vivo finding that the first-pass elimination of M1 was greater than that of DG. The kinetic analysis based on the distributed model showed that the influx (k'1) and efflux (k'2) rate constants for M1 were larger than those for DG, whereas no significant difference in the sequestration rate constant (k'3) was observed between the two ligands. Based on the concept proposed by Miyauchi et al. (J. Pharmacokinet. Biopharm., 15, 25 (1987)), it was suggested that the determinant factor of the hepatic intrinsic clearance was the influx clearance for both ligands, because the values of k'2 for each ligand were much smaller than the respective k'3 values. It was concluded that the higher plasma concentration of M1 after oral administration of RZ than that observed after administration of M1 is due to the fact that the hepatic uptake of DG is lower than that of M1.

General principals of drug absorption and bioavailability after the oral administration are discussed in each process of drug release and dissolution from the preparation, such as gastric emptying time and flow of drug in intestinal track, transport through intestinal mucosal microvilli, and the first pass effect of drug elimination in the liver. Character of lipid soluble vitamins in these absorption processes are elucidated with the low solubility in water and solubilization in bile acids micelle. Our studies on dl-alpha-tocopherol solubilization into micelles of bile acids and a detergent and the rat intestinal absorption by perfusion of the micellar solutions are introduced. Faster absorption of the tocopherol solubilized in bile acid micelles, than polysorbate 80, was observed in spite of the less solubilizing abilities of bile acids. Relatively high dependency on volume flow through the intestinal wall was observed in the absorption rate of tocopherol. The dependency is larger in bile acid micelles having smaller size than polysorbate 80 having large size.

The hepatic uptake clearances of 15 ligands with a wide range of permeabilities were determined in rats using two techniques: centrifugal filtration with isolated hepatocytes and the multiple indicator dilution (MID) method with isolated perfused livers. Some of the uptake clearance values were taken from the literature. Uptake clearance values obtained from isolated hepatocytes were extrapolated to that per gram liver (PSinf.cell), assuming that 1 g of liver has 1.3 x 10(8) cells. The values of PSinf.cell varied from approximately 0.1 to 72 (mL/min/g liver). The values of PSinf.cell were similar to those (PSinf.MID) determined by the MID method for ligands with uptake clearances below approximately 1 mL/min/g liver. However, for the ligands with larger uptake clearances, the PSinf.MID values were lower than the PSinf.cell values and appeared to reach an upper limit (approx. 15-20 mL/min/g liver). The PSinf.cell values of 1-propranolol, tetraphenylphosphonium (TPP+), and diazepam were 72, 43, and 22 mL/min/g liver, respectively, whereas their uptake clearances (PSinf.MID) determined by the MID method were 4 to 10 times lower. One of the possible mechanisms for this discrepancy is that an unstirred water layer, which may exist in Disse's space in isolated perfused livers (and probably under in vivo condition), limits the hepatic uptake rate of ligands with extremely high membrane permeabilities.

A physiologically based pharmacokinetic model was established to describe the hepatobiliary excretion process for ligands which are excreted into the bile without metabolic conversion. In this model, the following processes were taken into consideration: influx and efflux across the liver sinusoidal membrane, intracellular diffusion, and excretion across the canalicular membrane into the bile. The partial differential equation by which these processes were described was solved to obtain the Laplace transformed solution for the biliary excretion rate of ligands, based on the plasma concentration profiles as an input function. The time profiles for the biliary excretion rate of dibromosulfophthalein (DBSP; 6.80 mg/kg b.w.) or cefodizime (15.0 mg/kg b.w.) after i.v. bolus administration to rats were fitted to the solution using a nonlinear least-squares program based on a fast inverse Laplace transform (MULTI-FILT [Y. Yano et al., Chem. Pharm. Bull., 37, 1035 (1989)] to determine the permeability-surface area product across the plasma membrane as well as the apparent intracellular diffusion coefficient. DBSP and cefodizime were used as model compounds since these two ligands possess different binding characteristics for cytosol protein(s). Although both ligands are present predominantly in the cytosol, DBSP binds to intracellular protein(s) (such as ligandin) to a great extent whereas the protein binding of cefodizime is not so extensive. The fitted lines were superimposed on the experimental results.(ABSTRACT TRUNCATED AT 250 WORDS)

The significance of the binding to Na,K-ATPase in the tissue distribution of ouabain was previously documented (Harashima et al., Pharm. Res. 9:474-479, 1992). The purpose of this study was to obtain a kinetic model of ouabain tissue distribution. In most tissues, the ouabain concentration continued to rise after the termination of infusion (5 min), with the peak tissue concentration at approximately 20 min. This delay could not be explained by the rapid equilibrium model (RE model), nor could the kinetics of ouabain be explained by an RE model modified for saturable binding. Since ouabain binding to Na,K-ATPase is slow, the association and dissociation processes were incorporated into a model that can accurately fit the observed time courses of ouabain. The obtained binding parameters corresponded well with the observed values in the in vitro binding experiments, except for muscle. These results quantitatively support the role of the slow and saturable binding of ouabain to Na,K-ATPase in its tissue distribution.

To investigate the nonlinear kinetics in the hepatobiliary transport of rose bengal (RB), the time profiles of plasma concentration and biliary excretion rate after its i.v. administration at various doses were measured in rats. The total body clearance decreased remarkably with increased dose. The hepatic uptake clearance also showed a similar dose dependency, and saturation of hepatic uptake at least partly accounts for the dose-dependent change in total body clearance. The peak biliary excretion rate approached the transport maximum (approximately 150 nmol min-1 kg-1) with increased dose. To further clarify which process in RB hepatobiliary transport has nonlinearity, we analysed thus obtained data based on a three-compartment model. The hepatic uptake and sequestration rate constants decreased remarkably with increased dose. The initial hepatic uptake rates assessed from the plasma disappearance rate during the early phase fit well to the Michaelis-Menten equation with a saturable and a nonsaturable component. The maximum uptake velocity and Michaelis constant were 4.7 mumol min-1 kg-1 and 360 microM, respectively. That hepatic uptake has a much higher capacity (about 30 fold) than biliary excretion suggests that biliary excretion can be a rate-determining process in the overall hepatobiliary transport of RB. We conclude that the saturation of both hepatic uptake and biliary excretion could be the main causes for the nonlinear pharmacokinetics of hepatobiliary transport of RB.

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.

Circulating thrombomodulin is a novel endothelial cell marker, which may reflect the endothelial injury. Plasma levels of thrombomodulin were quantitated by an enzyme-linked immunosorbent assay (ELISA) in patients with hematological malignancies, liver disease, diabetes mellitus, collagen disease, thrombotic disease, and disseminated intravascular coagulation (DIC), and the thrombomodulin values were compared with those of von Willebrand factor antigen (vWf:Ag) and tissue-type plasminogen activator (t-PA) which are released from stimulated or damaged endothelial cells. The mean plasma concentrations of thrombomodulin in these disease states were elevated as compared with healthy subjects. A relatively high mean thrombomodulin level was observed in DIC, liver disease, and collagen disease. Abnormally high thrombomodulin values (greater than normal mean value + 3 SD) were found in 32.3% of patients with hematological malignancies, 57.7% of patients with liver disease, 39.3% of patients with diabetes mellitus, 30.0% of patients with collagen disease, 23.1% of patients with thrombotic disease, and 69.0% of patients with DIC. Plasma concentrations of both vWf:Ag and t-PA were also elevated in these patients. On the whole, the plasma thrombomodulin concentration was positively correlated with vWf:Ag (r = 0.441, P less than 0.001) and t-PA (r = 0.398, P less than 0.001). These findings indicate that the elevation of plasma thrombomodulin is frequently seen in a variety of diseases and circulating thrombomodulin is possibly useful for evaluating the endothelial damage in selected disease states.

In this paper, we offer experimental evidence of the rate-limiting diffusion of ligands through the unstirred water layer (UWL) as an explanation for the so-called albumin-mediated transport phenomenon. The relative membrane permeability of various ligands was first evaluated using isolated rat hepatocytes. Then, the effect of albumin on the uptake of ligands of a wide range of membrane permeabilities was examined using the perfused rat liver. The results were similar to those expected from the UWL model: ligands with high membrane permeability (warfarin, diazepam and taurocholate) clearly exhibited albumin-mediated transport, those with medium membrane permeability (tolbutamide and salicylate) showed less albumin-mediated transport, and ligand with low membrane permeability (cefodizime) did not show albumin-mediated transport. These results were explained by simulation studies of two separate cases based on the UWL model; one assuming the rapid equilibrium of ligand binding with albumin, and the other considering the slow dissociation of ligands from albumin. In light of these findings, we suggest that the rate-limiting diffusion through the UWL plays an important role in the so-called albumin-mediated transport phenomenon.

A rare association of congenital afibrinogenemia and hereditary protein C deficiency is described in a 37-year-old female who suffered from ischemic necrosis in the left first toe. The diagnosis of afibrinogenemia was assessed by the absence of fibrinogen in clotting and immunological assays. The diagnosis of hereditary heterozygous type I protein C deficiency was based on the evidence of proportional decreases of activity and antigen of plasma protein C in the propositus, her mother, and two maternal aunts.

Recent studies using bile canalicular membrane vesicles have suggested that P-glycoprotein may play a role in excreting some anticancer drugs from the liver to the bile. At steady state after a continuous single-pass perfusion of a tracer concentration of [3H]vincristine in the rat liver, the extraction ratio was approximately 0.6, and 70% of the extracted drug was excreted into the bile mostly in unchanged form. The liver/perfusate and bile/liver unbound concentration ratios obtained after correction for intracellular binding and the inside-negative membrane potentials and/or pH difference between the inside and outside of the cells, were approximately 2-3 and 160-280, respectively, suggesting a highly concentrated biliary excretion process. We also examined the effects of verapamil, a P-glycoprotein-related transport inhibitor in cancer cells, on the hepatobiliary transport of [3H]vincristine. Verapamil 50 microM in the perfusate caused a decrease in the biliary excretion rate of [3H]vincristine, whereas [14C]taurocholate (reference compound) remained constant. In contrast, the hepatic uptake rate of [3H]vincristine exhibited minimum reduction, suggesting that verapamil selectively inhibited the biliary excretion of [3H]vincristine at the canalicular membrane. The fact that verapamil had little effect on the initial velocity of [3H]vincristine uptake by isolated hepatocytes also supports the above findings. Since the effect of 150 microM verapamil in the perfusate was not selective for vincristine, the biliary excretion rates of both compounds ([3H]vincristine, [14C]taurocholate) were reduced by this concentration of verapamil. In conclusion, the concentrative excretion of vincristine into the bile and its selective inhibition by a moderate concentration of verapamil provide indirect evidence for the contribution of P-glycoprotein to the biliary excretion of vincristine in a perfused rat liver system.

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.

Although antiarrhythmic drugs are used to treat digitalis-induced cardiac disorders, some of these drugs have been reported to increase the serum digoxin concentration in patients, causing the severe side-effects. We have previously shown that many basic drugs including antiarrhythmic drugs inhibited the hepatic uptake of cardiac glycosides into isolated rat hepatocytes, which could be a cause for the increased serum digoxin concentration. The present study was designed to examine the mechanism of this inhibition using isolated rat sinusoidal plasma membrane vesicles. The effect of nine basic drugs (dipyridamole, nifedipine, verapamil, chlorpromazine, lidocaine, quinidine, ajmaline, disopyramide, and propranolol) on the uptake of ouabain was studied. Quinidine reduced the initial uptake rate of ouabain (30 s) while it did not change the uptake of ouabain in an equilibrium condition (30 min). Other basic drugs, such as verapamil, dipyridamole, and nifedipine also significantly reduced the initial uptake rate of ouabain. These basic drugs had no effect on the membrane fluidity. The inhibitory effects on the vesicular uptake were significantly correlated with the inhibitory effects on ouabain uptake by the isolated rat hepatocytes. These findings may suggest that the mechanism of the inhibition involves the inhibition of the transport process via the sinusoidal plasma membrane.

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.

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.

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.

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.

Uptake of organic anions into isolated rat hepatocytes was studied to examine their ATP dependency. In the presence of rotenone (0.2 microM), the initial velocity of the uptake (Vo) of dibromosulfophthalein (DBSP; 10 microM), 1-anilino-8-naphthalenesulfonate (ANS; 10 microM) and benzylpenicillin (PCG; 0.02 microM) was reduced to 60-70% of the control value, while that of bromosulfophthalein (BSP; 10 microM), rose bengal (RB; 10 microM) and bromophenol blue (BPB; 10 microM) was not affected. Furthermore, we examined the inhibitory effect of rotenone on the uptake at equilibrium of non-metabolizable ligands (DBSP, BPB and RB). The uptake of these ligands reached equilibrium at 30 min with a cel-to-medium concentration ratio (C/M ratio) of 75, 37 and 126, respectively. The C/M ratio at equilibrium of DBSP was reduced by rotenone to approx. 60% of the control value, while that of BPB and RB was not reduced. Other metabolic inhibitors such as sodium azide (10 mM) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 10 microM) also reduced the Vo of DBSP and PCG, while the uptake of BSP and RB was not reduced by these inhibitors. These results indicate that organic anions can be classified into two groups according to whether they are taken up by hepatocytes in an ATP-dependent manner, i.e., via active transport or in an ATP-independent manner, i.e., via facilitated diffusion. DBSP, PCG and ANS belong to the former group, whereas BSP, BPB and RB belong to the latter.

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.

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.

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.

Kinetic analysis of valproate (VPA) disposition in pregnant rats was performed. A dose-dependent saturable plasma elimination was observed in both the control and pregnant rats. Saturation was more remarkable in the pregnant rats and this was assumed to be due to the decreased hepatic extraction by metabolism. Pharmacokinetic parameters were calculated using a two-compartment open model with a Michaelis-Menten-type elimination process from the central compartment. In the pregnant rats, the calculated values of Km and Vmax decreased significantly, by one-half of those of the control rats, whereas no significant difference was observed in distribution rate constants (k12, k21) and volumes of distribution (V1, Vdss) between two groups of rats. Furthermore, the hepatic extraction of VPA in the control and pregnant rats was investigated in order to explain nonlinear pharmacokinetics of VPA. The values of the hepatic extraction ratio in the control and pregnant rats were 39.9 and 22.4% at the steady state concentration of approximately 20 micrograms/ml, respectively. The extraction ratios in the pregnant rats were lower than those of the control rats. This fact may be one of the reasons why the elimination of VPA in the pregnant rats is more susceptible to saturation.