Dual control of cytosolic metals by lysosomal transporters in lobster hepatopancreas.

This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal-thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of (65)Zn(2+) by lysosomal membrane vesicles (LMV) incubated in 1 mmol l(-1) glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l(-1) alpha-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l(-1) GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal (65)Zn(2+) influx as a function of zinc concentration, in vesicles containing 1 mmol l(-1) AKG and incubated in 1 mmol l(-1) GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.

Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis.

In addition to the better-known roles of the erythrocyte in the transport of oxygen and carbon dioxide, the concept that the red blood cell is involved in the transport and release of ATP has been evolving (J. Luthje, Blut 59, 367, 1989; G. R. Bergfeld and T. Forrester, Cardiovasc. Res. 26, 40, 1992; M. L. Ellsworth et al., Am. J. Physiol. 269, H2155, 1995; R. S. Sprague et al., Am. J. Physiol. 275, H1726, 1998). Membrane proteins involved in the release of ATP from erythrocytes now appear to include members of the ATP binding cassette (ABC) family (C. F. Higgins, Annu. Rev. Cell Biol. 8, 67, 1992; C. F. Higgins, Cell 82, 693, 1995). In addition to defining physiologically the presence of ABC proteins in RBCs, accumulating gel electrophoretic evidence suggests that the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance-associated protein (MRP1), respectively, constitute significant proteins in the red blood cell membrane. As such, this finding makes the mature erythrocyte compartment a major mammalian repository of these important ABC proteins. Because of its relative structural simplicity and ready accessibility, the erythrocyte offers an ideal system to explore details of the physiological functions of ABC proteins. Moreover, the presence of different ABC proteins in a single membrane implies that interaction among these proteins and with other membrane proteins may be the norm and not the exception in terms of modulation of their functions.

Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates: functional implications for adenosine triphosphate/drug cotransport in P-glycoprotein overexpressing tumor cells and in P-glycoprotein low-level expressing erythrocytes.

P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated with the expression of Pgp are in any way coupled. We have measured the stoichiometry of transport coupling between drug and ATP release. The drug and ATP transport that is inhibitable by the sulfonylurea compound, glyburide (P. E. Golstein, A. Boom, J. van Geffel, P. Jacobs, B. Masereel, and R. Beauwens, Pfluger's Arch. 437, 652, 1999), permits determination of the transport coupling ratio, which is close to 1:1. In view of this result, we asked whether ATP interacts directly with Pgp substrates. We show by measuring the movement of Pgp substrates in electric fields that ATP and drug movement are coupled. The results are compatible with the view that substrates for Pgp efflux are driven by the movement of ATP through electrostatic interaction and effective ATP-drug complex formation with net anionic character. This mechanism not only pertains to drug efflux from tumor cells overexpressing Pgp, but also provides a framework for understanding the role of erythrocytes in drug resistance. The erythrocyte consists of a membrane surrounding a millimolar pool of ATP. Mammalian RBCs have no nucleus or DNA drug/toxin targets. From the perspective of drug/ATP complex formation, the RBC serves as an important electrochemical sink for toxins. The presence in the erythrocyte membrane of approximately 100 Pgp copies per RBC provides a mechanism for eventual toxin clearance. The RBC transport of toxins permits their removal from sensitive structures and ultimate clearance from the organism via the liver and/or kidneys.

Doppler sonography findings associated with transjugular intrahepatic portosystemic shunt malfunction.

OBJECTIVE: Our purpose was to determine the overall accuracy of Doppler sonography and the accuracy of specific Doppler parameters associated with a compromised transjugular intrahepatic portosystemic shunt (TIPS). MATERIALS AND METHODS: For 43 patients who had undergone TIPS, 64 correlated sonogram-venogram paired examinations were analyzed. Sonographic parameters assessed included absolute velocities plus absolute and percentage changes in velocities measured in the main portal vein (MPV) and in several intrashunt locations (including peak and minimum velocity). Direction of flow and change in direction of flow in the left and right portal veins were also examined. TIPS malfunction was defined as any shunt with greater than or equal to 50% stenosis or any stenosis with a portosystemic gradient greater than 15 mm Hg. RESULTS: The prospective interpretation of the sonograms using all available parameters resulted in a sensitivity of 92% and a specificity of 72% for detecting TIPS malfunction. Peak shunt velocity (absolute velocity and velocity change), distal shunt velocity, MPV velocity (absolute velocity and percentage change in velocity), change in minimum shunt velocity, and direction of flow in branch portal veins were found to have statistically significant differences between normal and abnormal shunts. Sensitivities for these individual parameters ranged from 64% to 84%, and specificities ranged from 70% to 100%. When either the MPV velocity or the distal shunt velocity was abnormal, the sensitivity was 94%. When both parameters were abnormal, the specificity for detecting TIPS malfunction was 100%. CONCLUSION: Doppler sonography is a sensitive and relatively specific means of revealing TIPS malfunction. Accuracy depends on analysis of multiple sonographic parameters.

Stenosis of transjugular intrahepatic portosystemic shunts: presentation and management.

OBJECTIVE: The purpose of this study was to define the incidence, nature, and presentation of stenoses that develop in patients with transjugular intrahepatic portosystemic shunts (TIPS) and to assess the efficacy of treatment that prolongs shunt patency. MATERIALS AND METHODS: TIPS were successfully created in 108 patients over a 43-month period. Of the 93 patients with adequate radiologic or pathologic follow-up, 60 had no shunt problems and 33 developed shunt stenoses or occlusions. Follow-up of these 93 patients included sonography, venography, and/or pathologic confirmation. Presentations of stenoses, types of therapy, and patency after treatment were evaluated in all patients. RESULTS: In the cohort group, 35% of the patients had shunt problems (mean time to presentation, 7.4 months after TIPS). Forty stenoses and eight occlusions occurred in the 33 patients. Of the 48 shunt problems, 35 (73%) were detected with routine radiologic screening, 12 (25%) presented with recurrent symptoms, and one (2%) was confirmed by pathologic evaluation. Of the 33 patients with stenoses and occlusions, 21 had one reintervention, six had two reinterventions, three had three reinterventions, one had four reinterventions, and two received no therapy. These reinterventions included 30 restentings, 11 angioplasties, four new shunts, and one thrombolysis alone. Of the 31 primary reinterventions, 23 (74%) were restentings, six (19%) were angioplasties, and two patients received a new TIPS. Of the 10 secondary reinterventions, six were restentings, three were angioplasties, and one was a new TIPS. Of the four tertiary reinterventions, one was a restenting, two were angioplasties, and one was thrombolysis. Kaplan-Meier survival analysis revealed the primary patency of the shunt to be 67% at 6 months, 48% at 1 year, and 26% at 2 years. The primary-assisted patency of the shunt was 96% at 6 months and 87% at 3 years. The secondary patency was 99% at 1 year and 89% at 3 years. CONCLUSION: Stenoses are common after TIPS procedures and frequently can be detected on routine screening studies. Shunt revision can effectively extend the patency of TIPS. Restenting is generally required for hepatic vein stenoses. Angioplasty should be the first line of therapy for intrashunt stenoses, as only 44% of patients will require restenting.

Comparison of portal vein anatomy and bony anatomic landmarks.

PURPOSE: To determine if a relationship exists between the right portal trunk (RPT) and bony structures that might aid guidance of needle passes into the RPT during transjugular intrahepatic portosystemic shunt (TIPS) placement. MATERIALS AND METHODS: Sixty-two TIPS portal venograms were reviewed. The distance of the mid-RPT from the lateral margin of the vertebral column was measured and calculated as a fraction of the adjacent vertebral body width. The cephalocaudal height of the RPT was compared with that of the posterior ribs and rib spaces. The cephalocaudal height was evaluated with frequency distribution, and scattergram plots were used to determine the most common location of the mid-RPT relative to bony structures. The height and lateral position were analyzed in relation to clinical parameters to determine the effect of these parameters on RPT position. RESULTS: The mean distance of the mid-RPT from the lateral vertebral margin was 0.9 vertebral widths (range, 0.1-1.5). Fifty-six of 62 (90%) mid-RPTs were between 0.5 and 1.5 vertebral widths to the right of the lateral margin of the vertebrae. Fifty-four of 62 (87%) mid-RPTs were below the 10th and above the 12th ribs. Clinical factors did not affect RPT position. CONCLUSION: Bony landmarks provide an approximation of the mid-RPT location and may aid in TIPS placement.

Systemic venous enhancement patterns during dynamic abdominal CT.

In 25 patients we assessed the enhancement of abdominal venous structures during dynamic computed tomography (CT). The degree of venous enhancement demonstrated great variation. In six instances (out of 250 observations) a vessel was visually perceived as not enhancing and potentially thrombosed, including three gonadal veins. CT measurements were helpful in identifying enhancement, but were occasionally low enough that thrombosis remained a radiological consideration. The great variation in venous enhancement makes the diagnosis of thrombosis suspect, based on CT alone. Corroboration of this finding is suggested, when clinically relevant.

Safety of pulmonary angiography in the 1990s.

PURPOSE: To examine the safety of pulmonary angiography with low-osmolar contrast material and modern angiographic techniques and to analyze periprocedural complications with respect to potential predictors. PATIENTS AND METHODS: A retrospective review was conducted of data from 547 consecutive patients who underwent pulmonary angiography. Minor and major complications were analyzed by using several clinical parameters. RESULTS: There were five major (0.9%) and 26 minor complications (4.8%). Eleven of the 26 minor complications were contrast-induced nephrotoxicity. There were no periprocedural deaths. Patients with complications had an increased incidence of coexistent pulmonary morbidities and were of a poorer physical status according to the American Society of Anesthesiology criteria. Moderate to severe pulmonary hypertension was correlated with major complications. Age, volume of contrast material used, and presence of pulmonary embolism were not correlated with complications. CONCLUSION: Pulmonary angiography is a safe procedure with an acceptable complication rate. These findings should be considered in the selection of an imaging method for the diagnosis of pulmonary embolism.

Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.

The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5'-upstream region of the cytochrome P4501A1 gene (CYP1A1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (-782 to -843 bp) from a plasmid which contained the CYP1A1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5'-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site affinity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+ which contains the luciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.

Obstetric hemorrhage: prophylactic and emergency arterial catheterization and embolotherapy.

The role of arterial catheterization and embolotherapy was evaluated in 18 patients with postpartum hemorrhage or a risk of hemorrhage. Nine patients underwent emergency arterial catheterization for unanticipated postpartum hemorrhage due to uterine and vaginal tears and/or placental abnormalities. Bleeding was controlled with embolization with gelatin sponge in eight patients, while bleeding in one patient stopped spontaneously during angiography. Nine patients underwent prophylactic arterial catheterization before cesarean section or for abnormalities associated with risk of hemorrhage. Two subsequently underwent embolization before surgery, and embolization in two others was performed intraoperatively in response to serious bleeding. Bleeding in the other five was controlled by the usual surgical means. Arterial embolotherapy in these patients was an effective means of controlling postpartum hemorrhage. Prophylactic arterial catheterization has a role in patients with an increased risk for obstetric hemorrhage.

Color Doppler sonography of vascular malformations of the liver.

Nine cases of intrahepatic vascular malformations diagnosed using color Doppler sonography are described. These consisted of six cases of intrahepatic portal-hepatic venous shunts and three cases of arteriovenous fistulas. Among these is a case of multiple intrahepatic portal-systemic shunts. The sonographic findings and theories explaining the formation of vascular malformations in the liver are discussed.

Ossifying fibroma of sphenoid bone with coexistent mucocele: CT and MRI.

A case of a 26-year-old woman with decreasing vision in her right eye, diplopia, headache, and galactorrhea is presented. Both CT and MR studies showed a large sphenoid fibroosseous lesion that had a ground glass appearance on CT and low-to-intermediate proton density and low T2-weighted and low T1-weighted signal intensities on MR. After contrast medium administration, this process diffusely enhanced on CT and MR. There was also an expansile mass in the sphenoid sinus that had intermediate proton density, high T2-weighted and low T1-weighted signal intensities compared with brain. This mass did not enhance but had an intensely enhancing, uniformly thin rim. The pathologic diagnosis was ossifying fibroma with a sphenoid sinus mucocele. There are only isolated reports in the literature of benign fibrosseous lesions causing mucoceles. This association is reviewed as are the findings in this case.

CT findings after percutaneous biliary procedures.

A prospective study was performed to determine the frequency, type, and extent of abnormalities depicted with computed tomography (CT) after percutaneous biliary procedures (PBPs). Abdominal CT scans were obtained 24-72 hours after the PBP in 31 consecutive cases in 29 patients. Fifteen abnormalities were proved with CT in 14 patients (45%), as follows: subcapsular hematoma (two patients), subcapsular or perihepatic fluid collection (three patients), intrahepatic hematoma (three patients), nonspecific intrahepatic fluid collection (three patients), subcutaneous hematoma at the puncture site (one patient), free intraperitoneal air (one patient), intraperitoneal collection of contrast material (one patient), and inadvertent transxiphoid catheter tract (one patient). Only five of these patients had clinically apparent post-PBP complications that could be explained with CT findings. The 14 patients with positive CT findings required more needle passes (mean, 8.3 vs 4.6) during the PBP, had a difficult PBP more often (five patients [36%] vs four patients [27%]), and had more frequent placement of an internal-external drain (nine patients [64%]) than those with negative findings (eight patients [53%]). Positive findings on CT scans are common after a PBP and often are not associated with clinical symptoms.

Regulation of types I, III, and IV procollagen mRNA synthesis in glucocorticoid-mediated intestinal development.

Administration of dexamethasone (0.8 mg/kg) to 9-day-old rats once daily for 3 consecutive days caused precocious induction of adult specific disaccharidase activity in the small intestine. Maturation-specific disaccharidase activity was accompanied by decreased amounts of types I and III collagen and decreased procollagen type I and III mRNA levels. Conversely, type IV procollagen, fibronectin, and laminin amounts and their respective mRNA levels were increased. In vitro transcription of nuclei isolated from small intestine and colon of suckling rats indicated a decreased rate of synthesis of procollagen types I and III mRNAs and an increased rate of synthesis of procollagen type IV mRNAs and laminin mRNAs after dexamethasone treatment. The data suggest that glucocorticoids mediate a differential regulation of interstitial and basement membrane collagen gene expression in the developing rat intestine.

Glucocorticoids decrease the synthesis of type I procollagen mRNAs.

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone.

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.

Dexamethasone decreases the amounts of type I procollagen mRNAs in vivo and in fibroblast cell cultures.

Dexamethasone treatment of neonatal chicks resulted in a time- and dose-dependent selective decrease of skin collagen synthesis. Total RNA of chick skin was isolated and hybridized to the cloned cDNAs pCg54 for pro-alpha 1 (I) mRNA and pCg45 for pro-alpha 2(I) mRNA. RNA isolated from the total skin of chicks receiving various doses of dexamethasone had dose-related decreases of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs. The decrease of type I procollagen mRNAs for various doses of dexamethasone were similar to the decreases observed for collagen synthesis in vivo. Dexamethasone treatment of chick skin and chick lung fibroblasts resulted in a selective decrease of procollagen synthesis. A dose-related decrease of procollagen synthesis was observed with chick skin fibroblasts. Dexamethasone-treated chick skin and chick lung fibroblasts had decreased levels of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs as determined by solid support hybridization with pCg54 and pCg45. The dexamethasone-mediated decreases of type I procollagen mRNAs in skin fibroblasts and lung fibroblasts were similar to the decreases observed in procollagen synthesis.

Bleomycin-induced increase of collagen turnover in IMR-90 fibroblasts: an in vitro model of connective tissue restructuring during lung fibrosis.

Late-log-phase IMR-90 human fetal lung fibroblasts were incubated with bleomycin sulfate for 48 hr. The culture medium was removed and replaced with serum-free medium and [5-3H]proline. The cells were then incubated for increasing time intervals. The cells and culture medium were collected, and radioactive proline incorporated into collagen and noncollagen protein was determined. Intracellular collagen synthesis was selectively increased. Furthermore, polysomes isolated from bleomycin-treated cells synthesized significantly more collagen in the wheat germ lysate than did control polysomes. Prolyl hydroxylase activity was also increased significantly in the bleomycin-treated cells. Free and peptide-bound radioactive hydroxyproline in the cells and medium was greatly increased in bleomycin-treated cells, which indicates increased collagen degradation. The results demonstrate that, although collagen synthesis in lung fibroblasts is increased by bleomycin, the newly synthesized collagen is rapidly degraded in both the cell layer and the medium. This increased collagen degradation may be responsible for the remodeling which takes place during lung fibrosis.

Inhibition of collagen accumulation by glucocorticoids in rat lung after intratracheal bleomycin instillation.

Male Fischer 344 rats were given a single lung instillation of bleomycin sulfate (0.6 units/100 g). some animals were treated 24 hr after bleomycin administration with triamcinolone diacetate. Steroid treatment was continued on alternate days for 4 weeks. At the end of 4 weeks, the lungs of rats receiving bleomycin alone had two-fold increases of both prolyl hydroxylase activity and proteinaceous hydroxyproline as compared to control values. The lungs of bleomycin-treated rats which received 4 mg of triamcinolone diacetate per kg on alternate days had a 33% increase of prolyl hydroxylase activity and proteinaceous hydroxyproline content of bleomycin-treated animals receiving glucocorticoid (8 mg/kg) on alternate days were the same as control values. The results indicate that alternate day administration of the synthetic glucocorticoid triamcinolone diacetate blocks lung collagen accumulation following a single intratracheal dose of bleomycin to rats.