Identification of three cationic amino acid transporters in placental trophoblast: cloning, expression, and characterization of hCAT-1.

The concentrative transfer of amino acids from maternal to fetal blood is essential to fetal growth and metabolism. Cationic amino acids are transported across the placental microvillous and basal membranes by multiple pathways which act to mediate maternal/fetal transport. To identify the cationic amino acid transporters of human placenta, total RNA was harvested from cultured trophoblast and from the BeWo choriocarcinoma cell line, b30 clone, and used for reverse transcription (RT) and polymerase chain reaction (PCR). Primers based on published sequences identified expression of mRNAs for hCATs-1, -2B, and -4. RT-PCR yielded a 2.1 kb hCAT-1 cDNA which was cloned. hCAT-1 cRNA injection into Xenopus laevis oocytes stimulated saturable lysine uptake (K(m) approximately 100 microM). In the presence of Na(+), uptake was inhibited by leucine, homoserine, and alanine but not by valine and glutamate. These transport characteristics are comparable to those of system y(+) in placental basal membrane, but differ from those of the same system in microvillous membrane. The identification, cloning, and characterization of multiple human placental cationic amino acid transporters has the potential to facilitate molecular investigation of transport by the maternal- and fetal-facing membranes of placental trophoblast and increase understanding of the mechanism of transplacental amino acid transfer.

Isotope dilution gas chromatographic-mass spectrometric measurement of tricyclic antidepressant drugs. Utility of the 4-carbethoxyhexafluorobutyryl derivatives of secondary amines.

Stable isotope dilution gas chromatographic-mass spectrometric (GC-MS) measurement of tricyclic antidepressants (TCA) is a useful alternative to high-performance liquid chromatography (HPLC) methods when interfering substances prevent accurate quantitation by HPLC. For satisfactory GC-MS analysis, secondary amine TCA must be derivatized. Commonly employed trifluoroacetyl and heptafluorobutyryl derivatives are relatively unstable and cause rapid deterioration of capillary GC columns. Therefore we examined 4-carbethoxyhexafluorobutyryl chloride (CHFB-CI) as an alternative derivatizing agent and developed a stable isotope dilution GC-MS method employing ring-labeled [2H4]-desipramine and [2H4]-imipramine internal standards, which permits measurement of desipramine, nortriptyline, imipramine, and amitriptyline in plasma samples containing one or all of these analytes. The GC-MS assay is linear for each analyte from the lower limit of quantitation (25 ng/mL) up to 1500 ng/mL and correlates well with HPLC measurements. The GC-MS analytic coefficient of variation was 9.7 +/- 1.3% for all analytes considered together. Although interferences are observed in the HPLC assay, thioridazine, perphenazine, cyclobenzaprine, and norcyclobenzaprine do not interfere with GC-MS measurements of the TCA examined here. The stability of the CHFB derivative of secondary amine TCA was found to be superior to that of the trifluoroacetyl derivatives of these compounds.

Sodium-independent lysine uptake by the BeWo choriocarcinoma cell line.

Transport of L-lysine by a cultured placental trophoblast cell line was investigated by characterization of L-[3H]lysine uptake. In the mononuclear form of the BeWo clone b30 choriocarcinoma cell, at least two sodium-independent systems are present. Concentration dependence data were fitted by a two system model with Km values (+/- s.e.) of 2 +/- 0.7 and 94 +/- 31 microM and Vmax values (+/- s.e.) of 0.7 +/- 0.3 and 25 +/- 6.0 nM/mg DNA/min. A portion of sodium-independent uptake was inhibited by the sulphydryl modifying reagent N-ethylmaleimide (NEM). Following NEM treatment, the data were fitted by a single system with Km = 10 +/- 2 microM AND Vmax = 5.1 +/- 0.8 nM/mg DNA/min. In the absence of sodium, NEM-resistant uptake was sensitively inhibited by leucine whereas NEM-sensitive uptake was not inhibited by leucine. It is concluded that like placental basal membrane, the mononuclear BeWo cell possesses two sodium-independent L-lysine transport systems. The high-capacity, NEM-sensitive, leucine-insensitive system resembles the widespread system y+. The high-affinity, NEM-resistant, leucine-sensitive system resembles system b(0,+).

Comparison between the CEDIA and EMIT II immunoassays for the determination of benzodiazepines.

We evaluated a new, qualitative immunoassay for benzodiazepines in urine using CEDIA technology on the Hitachi 747 and compared its performance to an immunoassay using EMIT II methodology on the same instrument. A total of 500 urine samples received for routine drug screen analysis were prospectively examined for benzodiazepines by both methods. Samples producing positive results by either immunoassay method were analyzed by gas chromatography-mass spectrometry (GC-MS). Available medical records were reviewed for patients whose samples produced discrepant immunoassay results or that were positive in both immunoassays but negative by GC-MS. Samples that produced negative results in both immunoassays were not subjected to GC-MS analysis. Therefore, identification of an immunoassay result as a false negative only occurred when the sample produced a positive value in only one of the two immunoassays and was confirmed as positive by either GC-MS or medical record review. Following initial immunoassay screening and confirmation by GC-MS, a medical record review and reanalysis of GC-MS data was performed. After this in-depth analysis of the data, the CEDIA method produced 60 true-positives, 7 false positives and no false negatives. The EMIT II method produced 47 true positives, 1 fase positive and 13 false negatives. These differences appear to be due to the CEDIA assay being more sensitive for detection of lorazepam.

Measurement of plasma ibuprofen by gas chromatography-mass spectrometry.

A gas-chromatography-mass spectrometry (GC-MS) method for the determination of plasma ibuprofen was developed. Plasma samples from cystic fibrosis (CF) patients receiving high-dose ibuprofen therapy were analyzed by GC-MS and the result compared to analysis by high-performance liquid chromatography (reference method). Analysis of ibuprofen was sensitive to at least 5 mg/L, and the method was linear to 200 mg/L. Within-run variations of plasma samples were 4.6% (131.7 +/- 6.0 mg/L) and 5.4% (44.4 +/- 2.4 mg/L), respectively. The between-run variation was 9.3% (45.4 +/- 4.2 mg/L) and 7.4% (88.0 +/- 6.5 mg/L). This method is suited for routine clinical use for the monitoring of plasma ibuprofen levels in treatment of CF. It may be particularly applicable in pediatric laboratories, which are likely to possess GC-MS capability.

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity.

pp60c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443-23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependent activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+ cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+ cells, but the effect was smaller in PTPase+ cells. In an in vitro assay, hormone-stimulated pp60c-src autophosphorylation from PTPase+ cells was decreased 64 +/- 22%, and substrate phosphorylation by pp60c-src was reduced 54 +/- 16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, an in vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition, in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was an in vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.

Functional insulin and insulin-like growth factor-1 receptors are preferentially expressed in multiple myeloma cell lines as compared to B-lymphoblastoid cell lines.

While IGF-1 plays a role in early B-cell development, little is known of insulin and insulin-like growth factor-1 (IGF-1) action in post-marrow B-cells. Recently, our laboratory demonstrated that mouse and human multiple myeloma (MM) cell lines possess functional insulin receptors (IRs) and IGF-1 receptors (IGF-1Rs). In this study, we show that responsiveness to insulin and IGF-1 is more developed in human MM cell lines than in human B-lymphoblastoid cell lines. Two human MM cell lines (U266 and RPMI 8226) were compared to three B-lymphoblastoid cell lines [Epstein-Barr virus immortalized B-cells (EBV), a Burkitt lymphoma cell line (Ramos), and a non-EBV lymphoblastoid cell line (HS Sultan)]. Surface IR and IGF-1R expression, measured by flow cytometry, demonstrated that the MM cell lines expressed more IRs and IGF-1Rs than did the EBV, Ramos, or HS Sultan cell lines. In vitro receptor kinase activity of affinity-purified receptors showed that the MM cells had more phosphorylated receptors than did the EBV, Ramos, or HS Sultan cells. Intracellular receptor signaling was also markedly different between the two cell groups. Whole cell phosphorylation studies showed that MM cells possessed not only hormone-dependent receptor autophosphorylation (M(r) 97,000) but also substrate phosphorylation (M(r) 185,000; 60,000). The lymphoblastoid cells, while demonstrating receptor autophosphorylation (IR autophosphorylation in the EBV cell line at 200 nM hormone was similar to MM receptor phosphorylation at 2 nM), lacked hormone-responsive substrates. The MM cell lines contained significantly more hormone-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity than the B-lymphoblastoid cell lines. In the MM cells, PI 3-kinase was activated by at least 10-fold, but, in the B-lymphoblastoid cell lines, it was activated by no more than 2-fold. Hormone-responsive glucose metabolism was also greater in the MM cell lines. In the U266 cells, insulin increased lactate production 62 +/- 9 and 101 +/- 12% (mean +/- SE) at concentrations of 2 nM and 200 nM, respectively. IGF-1 produced 72 +/- 9 and 99 +/- 13% increases at similar concentrations. In the 8226 cells, insulin increased lactate production 4 +/- 4 and 36 +/- 15% at 2 and 200 nM, respectively. IGF-1 produced a 13 +/- 6 and 70 +/- 18% increase. In the EBV and Ramos cells, neither hormone increased lactate production by more than 10 +/- 3%.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of phosphatidylinositol-3-kinase by platelet-derived growth factor and insulin-like growth factor-1 is inhibited by a transmembrane phosphotyrosine phosphatase.

Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit hormone-dependent tyrosine phosphorylation and mitogenesis (Mooney, R. A., Freund, G. G., Way, B. A., and Bordwell, K. L. (1992) J. Biol. Chem. 267, 23443-23446). Here the impact of PTPase expression on insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor- (PDGF) dependent activation of PI-3-K was investigated. In PTPase+ cells, IGF-1 and PDGF-dependent PI-3-K activity in antiphosphotyrosine immunoprecipitates was decreased by 62 +/- 13 and 46 +/- 17%, respectively, compared to control cells. Similar decreases in PI-3-K activity associated with anti-PDGF receptor and anti-insulin receptor substrate-1 (IRS-1) immunoprecipitates were also observed. Association of PI-3-K with the hormone-activated PDGF receptor decreased approximately 55%, paralleling its loss of activation in PTPase+ cells. Tyrosine phosphorylation of the 85-kDa subunit of PI-3-K was also inhibited. Similarly, IGF-1 dependent tyrosine phosphorylation of IRS-1 was decreased by 45%, and its association with PI-3-K was decreased by 65% in PTPase+ cells. Finally, PDGF-dependent tyrosine phosphorylation of phospholipase C-gamma 1 and GTPase-activating protein was reduced by 60-70% in the PTPase+ cells as was tyrosine phosphorylation of the PDGF receptor associated with these proteins. In summary, expression of a transmembrane PTPase decreased hormone-dependent PI-3-K activation, tyrosine phosphorylation of receptor substrates, and their association with signaling complexes. These data support a role for transmembrane PTPases in the regulation of receptor signal transduction pathways.

Vascular morphometry of KHT and RIF-1 murine sarcomas.

Although tumor oxygenation and vascular structure have been studied extensively, previous work has most often been qualitative in nature. To clarify underlying physiological mechanisms, a more quantitative approach is needed. The current work considers two murine tumor lines of differing radiobiological hypoxic fraction (HF), the RIF-1 and the KHT fibrosarcomas. Following intravascular injection of India ink, histological sections were prepared and quantitated in terms of anatomical blood vessel-tumor cell distance distributions and vessel diameters. Vessel diameters increased with increasing tumor volume for RIF-1 tumors, while not changing significantly for the KHT. The fraction of the tumor within a given distance of the nearest blood vessel, varied as a function of: (i) tumor line, (ii) distance from tumor surface, and (iii) tumor volume. For small-volume tumors, intertumor differences in vessel-tumor cell distances correlated with differences in radiobiological HF, while for large-volume tumors, vessel-tumor cell differences were not significantly different between tumor lines. Combining current findings with previously determined intravascular oxyhemoglobin distributions in the same two tumor lines, it was apparent that not only is a substantial portion of the tumor volume beyond the expected oxygen diffusion limits of blood vessels, but, in addition, a relatively low percentage of these vessels is capable of supplying oxygen.

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.

Immunolocalization, quantitation and cellular heterogeneity of apolipoprotein B in rat hepatocytes.

Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.

The inflammatory response to endotoxin.

A typical inflammatory response resulted from the intravenous injection of endotoxin (E. coli) into living rabbits. Each rabbit was studied at three levels: the microvasculature and supporting tissue in the ear chamber was observed microscopically (up to X200) before, during, and at regular intervals following the injection of endotoxin; leucocyte and platelet counts were made periodically throughout each experiment; and tissue samples for histological study were obtained from each rabbit prior to death. The animal was anaesthetised before histological samples were secured. Within minutes after the intravenous injection of endotoxin, leucocytes were observed sticking to the endothelial cells lining the venules and the arterioles. Emboli appeared in the microcirculation within 10 min. Swelling of the microvascular endothelial cells was evident at 1 hr; oedema and extravasation of the cellular elements followed. The rectal temperature and leucocyte and platelet counts all fell within 10 min. of endotoxin. Histological examination of tissue from the ear chamber and visceral organs showed inflammatory changes. Congestion of the microvasculature, swelling of the endothelial cells, and margination and migration of neutrophils were common histological features in all organs. The earliest cells affected appeared to be the leukocyte and platelet.