Human Liver Membranes Research Articles

Structure-Property Relationship Studies of Influenza A Virus AM2-S31N Proton Channel Blockers.

Majority of current circulating influenza A viruses carry the S31N mutation in their M2 genes, rendering AM2-S31N as a high profile antiviral drug target. With our continuous interest in developing AM2-S31N channel blockers as novel antivirals targeting both oseltamivir-sensitive and -resistant influenza A viruses, we report herein the structure-property relationship studies of AM2-S31N inhibitors. The goal was to identify lead compounds with improved microsomal stability and membrane permeability. Two lead compounds, 10d and 10e, were found to have high mouse and human liver microsomal stability (T 1/2 > 145 min) and membrane permeability (>200 nm/s). Both compounds also inhibit both currently circulating oseltamivir-sensitive and -resistant human influenza A viruses (H1N1 and H3N2) with EC50 values ranging from 0.4 to 2.8 μM and a selectivity index of >100. We also showed for the first time that AM2-S31N channel blockers such as 10e inhibited influenza virus replication at both low and high multiply of infection (102-106 pfu/mL) and the inhibition was not cell-type dependent. Overall, these studies have identified two promising lead candidates for further development as antiviral drugs against drug-resistant influenza A viruses.

Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants

BackgroundHepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. MethodsProtein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. ResultsWe analyzed 36 fetal (median GA 23.4 weeks [range 15.3–41.3]), 12 premature newborn (GA 30.2 weeks [24.9–36.7], PNA 1.0 weeks [0.14–11.4]), 10 term newborn (GA 40.0 weeks [39.7–41.3], PNA 3.9 weeks [0.3–18.1]), 4 pediatric (PNA 4.1 years [1.1–7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. Discussion and conclusionVarious developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge.

The endogenous production of hydrogen sulphide in intrauterine tissues

BackgroundHydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.MethodsThe production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated.ResultsThe order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion.ConclusionRat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy.

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 μCi of [ 14C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 μmol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min·mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K m of 38 ± 25 and 45 ± 15 μmol/L, whereas the K m for retigabine N-glucuronidation by human liver microsomal fractions was 145 ± 39 μmol/L. Furthermore, a V max of 1.2 ± 0.3 (nmol/[min·mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine– N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 μmol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of 14C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids

Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3′-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4′-position to yield eriodictyol and quercetin, respectively. Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and α-naphthoflavone. Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2. CYP2C9 catalyzed the 4′-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids. The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.

Autoimmune reactivity of sera to hepatocyte plasma membrane in type 1 autoimmune hepatitis.

Type 1 autoimmune hepatitis (AIH-1) is an organ-specific autoimmune liver disease for which no tissue-specific autoantigen has yet been identified. We examined the reactivity by sensitive immunoblotting with enhanced chemiluminescence (IB-ECL) of 43 sera from patients with AIH-1 and 182 sera from patients with other diseases on hepatocyte plasma membrane derived from rat or human liver (RHPM, HHPM) and separated by aqueous two-phase partition. The sera studied were from patients with AIH-1, primary biliary cirrhosis, chronic viral hepatitis, and systemic lupus erythematosus (SLE); and from normal subjects. Specificity of reactivity by IB-ECL was sought: (i) by testing sera on human or rat liver membrane; (ii) by testing sera on liver or kidney membrane; (iii) by serial titration of reactive sera; and (iv) by testing reactive sera from AIH-1 before and after successful treatment with prednisolone. The results were that in AIH-1 there were multiple reactive components which were not species-specific, since they were detected with both RHPM and HHPM, but were mostly tissue-specific for liver. There was no significant correlation between antinuclear antibodies (ANA) titer and the frequencies of sera reactivities against RHPM. Most of these reactive components were demonstrable at a lesser frequency in other liver diseases and in SLE. There was a striking decrease in reactivity by IB-ECL of AIH-1 sera with liver membrane after clinical remission, further suggesting that differences between AIH-1 and other inflammatory liver diseases and SLE are predominantly quantitative rather than qualitative. However, our study did point to candidate liver membrane antigens with molecular sizes of 136, 116, 81, and 49 kDa, additional to components previously described by others. The molecular identification of these prominent reactants with AIH-1 sera could prove informative for ascertaining pathogenesis.

Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.

Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.

Isolation and Partial Characterisation of Insulin-Mimetic Inositol Phosphoglycans from Human Liver

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolpyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose–oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C fromBacillus thuringiensisand were resistant to glycosylphosphatidylinositol-specific phospholipase C fromTrypanosoma brucei.These data indicate that IPG molecules with insulin-like biological activities are present in human liver.

Glycosyl-phosphatidylinositol-phospholipase Type D: A Possible Candidate for the Generation of Second Messengers

Membrane associated glycosyl-phosphatidylinositols have been shown to be the precursors of inositol phosphoglycan second messengers. Extraction of human liver membranes and purification by serial thin layer chromatography revealed three glycolipids which co-migrated with glycosyl-phosphatidylinositol from rat liver. These lipidic fractions were partially sensitive to treatment with nitrous acid and to hydrolysis by glycosyl-phosphatidylinositol-specific phospholipase D from bovine serum. In parallel, glycosyl-phosphatidylinositol isolated from rat liver was found to be a substrate for the enzyme generating a biologically active inositol phosphoglycan species (determined by measuring inhibition of protein kinase A activity and stimulation of cell proliferation within the chicken embryo cochleovestibular ganglion). This molecule was recognised by an anti-inositol phosphoglycan antibody. Hence, we propose that glycosyl-phosphatidylinositol-specific phospholipase D could be implicated in cellular signalling.

Metabolism of vasopressin, oxytocin and their analogues [Mpa1, d-Arg8]-vasopressin (dDAVP) and [Mpa1, d-Tyr(Et)2, Thr4, Orn8]-oxytocin (antocin) in human kidney and liver homogenates

Information regarding the metabolic fate of the neurohypophyseal hormones arginine-vasopressin (AVP), oxytocin (OT) and their analogues in man is practically non-existent. The aim of the present study was to investigate the stability of oxytocin, vasopressin and their analogues dDAVP and [Mpa1-d-Tyr2(Et), Thr4, Orn8]-oxytocin (antocin) in human renal microvilli brush border membranes and in human liver membranes. After incubation the extent of degradation of the peptides was determined by reversed phase high-performance liquid chromatography (HPLC). The degradation of both AVP and OT was rapid in the presence of glutathione and human renal microvilli membranes. AVP, as well as dDAVP, was stable when incubated with microvilli membranes without glutathione, while OT was metabolized. The metabolization of the oxytocin analogue, antocin, also varied with the presence of glutathione. While in the absence of glutathione a more lipophilic peak eluted, a more hydrophilic peak was observed with glutathione on HPLC. The lipophilic peak was found to coelute with the truncated analogue [Mpa1, d-Tyr2 (Et), Thr4, desOrn8, Gly9]-oxytocin. No degradation occurred when the peptides were incubated with liver membranes. However, when using crude, unpurified liver homogenate degradation occurred for all peptides except antocin. The degradation of AVP in the human unpurified liver homogenate was as rapid as in the renal microvilli membranes. Similarly, OT was more rapidly degraded in human kidney microvilli membranes in the presence of glutathione than in the human crude liver homogenate, when using equal amounts of protein in the incubations. Thus, the present investigation indicates the existence of two possible metabolic pathways, in kidney microvilli, one for OT, which did not require the presence of reduced glutathione, and one for AVP, which required the presence of reduced glutathione. Liver degradation, on the other hand, requires the hepatocytes.

Molecular and physiological properties of clonidine-displacing substance.

An endogenous small molecular mass compound, termed clonidine-displacing substance (CDS), has been isolated and purified from bovine brain. The estimated level of CDS in bovine brain is 400-1,000 units/wet brain, with 1 unit of activity calculated to be approximately 1-2 ng. It is present in human serum, urine, and cerebrospinal fluid. The isolation procedure consists of initial aqueous and methanolic extractions followed by a series of HPLC chromatography steps (reverse phase and TSK sizing columns). The reverse-phase chromatography of CDS extracted under identical conditions from bovine brain and human serum show similar retention times. The final chromatography step gives a single active peak with a distinct ultraviolet spectrum, a single molecular peak m/z 587.8 +/- 2 in plasma desorption mass spectrometry (PDMS), and a unique pharmacological and physiological profile. Clonidine-displacing substance does not partition into organic solvents and it is ninhydrin and fluorescamine negative. All of these molecular properties clearly distinguish CDS from agmatine, an endogenous 130-dalton compound of far greater abundance which displays lower affinity for p-aminoclonidine-labeled sites in rat brain membranes. The ultraviolet spectrum of CDS consists of two aromatic peaks at 224 and 276 nm, whereas agmatine is an aliphatic substance with no ultraviolet absorbance. Like many antihypertensive drugs of the guanidine and imidazoline family of compounds, CDS recognizes alpha 2-adrenergic receptors, clonidine sites (IR-I1), and imidazoline sites (IR-I2). A good correlation exists between the affinities of various imidazoline/guanidine type ligands for IR-I2 in both human placenta and rat liver membranes which can be accurately determined because both tissues lack IR-I1 and alpha 2-adrenergic receptors. There is no correlation in the affinities of these ligands for IR-I2 of human-placental versus alpha 2-adrenergic receptors of human platelets. By uncovering the role of CDS in the central nervous system we will be able to understand the coupling of IRs to neurotransmission and, in turn, to changes in arterial pressure.

Characterization of the human liver α1-adrenoceptors: predominance of the α1A subtype

The α 1-adrenoceptor subtype present in human liver membranes was studied using radioligand binding techniques. [ 3H]Prazosin binding was rapid, saturable and reversible. A kinetically derived K d of 0.22 nM was obtained. Rosenthal analysis of saturation isotherms indicated a single class of binding sites with a K d of 0.47 nM and a B max of 70 fmol/mg of protein. Membrane preincubation with chloroethylclonidine markedly decreased total binding (62% decrease) without altering the K d for the radioligand. Binding competition experiments were performed and the order of potency for agonists was: oxymetazoline > epinephrine ⪰ norepinephrine > methoxamine . The binding affinity for epinephrine was modulated by the GTP analogue guanosine-5′-(β, γ-imido)triphosphate. For antagonists the potency order was: WB4101 ⪰ prazosin ⪰ (+)-niguldipine = 5-methylurapidil ⪰ benoxathian ⪰ phentolamine . The pharmacological profile of the [ 3H]prazosin binding sites of human liver membranes suggests that α 1A-adrenoceptors predominate (75%–85% of the α 1-adrenoceptors) in this tissue.

The Hep G2 cell line in the study of growth hormone receptor/ binding protein

This study identifies specific, high affinity GH-receptors (GH-R) in human hepatoma Hep G2 cells. The binding characteristics of GH-R in the Hep G2 cells are similar to those of human liver membranes, such as the high specificity for hGH, the binding affinity (K a = 1.7 ± 0.5 × 10 9 M −1) and the molecular weight of the membrane bound GH-R (apparent 125,000 and 71,000). In addition, lower molecular weight forms (∼ 94,000 and ∼ 58,000) were identified as GH-binding protein (GH-BP) in Hep G2 conditioned medium, or following incubation of Hep G2 cells, in the presence of 10 mM N-ethylmaleimide for 90 min at 30°C; the latter are presumed to be shed by a proteolytic cleavage of the GH-R. Exposure of Hep G2 cells to physiologic concentrations of hGH resulted in a concentration-dependent increase in 3H-thymidine incorporation, up to 48.4 ± 7.9% above control. In summary, the demonstration of specific, high affinity GH-R in Hep G2 cells, as well as shedding of GH-BP, suggest these cells may provide a homologous human system to study the receptor-effector interrelationship of hGH and to further our understanding of hepatocyte production of soluble GH-BP.

Characterization of vasoactive intestinal peptide receptors in human liver

The stoichiometric, pharmacological and molecular properties of vasoactive intestinal peptide (VIP) receptors have been analyzed in human liver membranes and compared in parallel with those in rat liver membranes. The binding of [ 125I]VIP was rapid, saturable and specific. The stoichometric data indicated the presence of two classes of binding sites in both human and rat liver membranes with K d values of 0.22 (human) and 0.20 (rat) nM for the high-affinity site, and 27.3 (human) and 3.6 (rat) nM for the low-affinity site. Tracer binding was displaced by structurally related peptides with an order of potency: VIP = PACAP-27 > helodermin > secretin in human liver, and VIP = PACAP-27 = helodermin > secretin in rat liver. GTP inhibited [ 125I]VIP binding in a dose-dependent manner suggesting the involvement of a G protein in the signal transduction pathway. Cross-linking experiments revealed an apparent molecular mass for the VIP-receptor complex that was 67500 ± 2700 and 50500 ± 900 in human and rat preparations, respectively. VIP receptors were functional, since VIP stimulated adenylyl cyclase activity in a dose dependent manner with similar efficacy but different potency in human (ED 50 = 1.2 nM) and rat (ED 50 = 5.8 nM) liver membranes.

P13 Absorption enhancement of hydrophilic compounds by racemic verapamil after rectal administration in rats

Information regarding the metabolic fate of the neurohypophyseal hormones arginine-vasopressin (AVP), oxytocin (OT) and their analogues in man is practically non-existent. The aim of the present study was to investigate the stability of oxytocin, vasopressin and their analogues dDAVP and [Mpa1-d-Tyr2(Et), Thr4, Orn8]-oxytocin (antocin) in human renal microvilli brush border membranes and in human liver membranes. After incubation the extent of degradation of the peptides was determined by reversed phase high-performance liquid chromatography (HPLC). The degradation of both AVP and OT was rapid in the presence of glutathione and human renal microvilli membranes. AVP, as well as dDAVP, was stable when incubated with microvilli membranes without glutathione, while OT was metabolized. The metabolization of the oxytocin analogue, antocin, also varied with the presence of glutathione. While in the absence of glutathione a more lipophilic peak eluted, a more hydrophilic peak was observed with glutathione on HPLC. The lipophilic peak was found to coelute with the truncated analogue [Mpa1, d-Tyr2 (Et), Thr4, desOrn8, Gly9]-oxytocin. No degradation occurred when the peptides were incubated with liver membranes. However, when using crude, unpurified liver homogenate degradation occurred for all peptides except antocin. The degradation of AVP in the human unpurified liver homogenate was as rapid as in the renal microvilli membranes. Similarly, OT was more rapidly degraded in human kidney microvilli membranes in the presence of glutathione than in the human crude liver homogenate, when using equal amounts of protein in the incubations. Thus, the present investigation indicates the existence of two possible metabolic pathways, in kidney microvilli, one for OT, which did not require the presence of reduced glutathione, and one for AVP, which required the presence of reduced glutathione. Liver degradation, on the other hand, requires the hepatocytes.

Binding of ( 3H) SR 49059, a Potent Nonpeptide Vasopressin V 1a Antagonist, to Rat and Human Liver Membranes

The new potent and selective nonpeptide vasopressin V 1a antagonist, SR 49059, was tritiated and used for the characterization of rat and human liver AVP V 1a receptors. Binding of [ 3H] SR 49059 was time-dependent, reversible and saturable. A single class of high affinity binding sites was identified with K d values of 0.63 ± 0.13 and 2.95 ± 0.64 nM, in rat and human liver membranes, respectively. The maximal binding capacity (B max) was about 7 times higher in rat than in human liver preparations. The relative potencies of several AVP/oxytocin agonists or antagonists to inhibit [ 3H] SR 49059 binding confirmed that this ligand labeled a homogenous population of sites with the expected AVP V 1a profile. Furthermore, [ 3H] SR 49059 or unlabeled SR 49059 displayed only slight species differences between rat and human V 1a receptors, whereas OPC-21268, another nonpeptide V 1a antagonist, exhibited a high species-related potency with more than 500 fold higher affinity for rat than for human liver V 1a receptors. Thus, [ 3H] SR 49059 is the first nonpeptide AVP V 1a ligand reported having highly specific activity, stability, specificity and affinity. This makes it a suitable probe for labeling AVP V 1a receptors in rat and also in human tissues.

T Cell Clones Generated from Patients with Type 1 Diabetes Using Interleukin-2 Proliferate to Human Islet Antigens

T lymphocytes are implicated in the pathogenesis of Type 1 (insulin dependent) diabetes. Activated T lymphocytes expressing IL-2 receptors are found at increased levels in the peripheral blood in the prediabetic period, at diagnosis and for several months after the onset of the disease, but their role in the pathogenesis of the disease is not known. We have used co-culture of peripheral blood lymphocytes with IL-2 alone to selectively generate T cell clones from the in vivo activated T cell population, and examined the phenotype and antigen specificity of the clones derived. From 3 patients with newly-diagnosed Type 1 diabetes 184 clones were generated, the majority of which (39%) were CD4+TCR alpha beta+, whilst 31% were CD8+TCR alpha beta+. From 2 healthy control subjects 90 clones were obtained, of which 62% were CD4+TCR alpha beta+ and 33% were CD8+TCR alpha beta+. Antigen specificity was examined in 46 clones from the patients and 44 from the control subjects in proliferation assays, using as antigens homogenate of human islets of Langerhans, human islet membrane preparation and human liver membrane preparation. Three clones (all CD4+TCR alpha beta+) from the patients, but none from the control subjects, proliferated in a dose dependent fashion in response to stimulation with human islet homogenate presented by autologous APCs, but to neither of the other autoantigen preparations. Our results demonstrate that a relatively high proportion (7%) of T lymphocytes activated in vivo recognise human islet antigens, indicating that they may have a role in the pathogenesis of the disease.

Significance of anti-eye muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.

To investigate the prevalence of antibody against rat eye muscle membrane antigen, as determined from SDS-polyacrylamide gel electrophoresis and western blotting, in sera from patients with thyroid-associated ophthalmopathy (TAO), we quantitatively analyzed the binding activity with a rat eye muscle membrane 64 kDa protein using chromato-scanner. Eye muscle antibody activity was expressed as ratio of density of the 64 kDa band to that at 66 kDa found with all normal sera and phosphate buffered saline. The mean (+/- SD) eye muscle antibody activity was 2.7 +/- 2.7 in TAO (P < 0.01 v.s. normal), 1.5 +/- 1.7 in Graves' disease without evident eye disease, 1.6 +/- 2.5 in Hashimoto's thyroiditis and 0.45 +/- 0.26 in normal subjects. A positive band at 64 kDa was found in 71% of patients with TAO, 36% of those of Graves' disease without evident eye disease and in 35% of patients with Hashimoto's thyroiditis without eye disease. The prevalence of this antibody activity tended to correlate to the severity of ophthalmopathy. Furthermore, the level of eye muscle antibody activity decreased in parallel with the improvement of eye signs in two patients. Sera reactive with rat eye muscle membrane 64 kDa protein reacted also with a human eye muscle membrane 64 kDa protein but not with human thyroid, liver, spleen or pancreas membrane preparations. In conclusion, antibody to rat eye muscle membrane 64 kDa protein is present in TAO and may be a useful clinical marker of ophthalmopathy.

Characterization of the binding of plasminogen activators to plasma membranes from human liver.

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

Localization of the growth hormone receptor, identified by immunocytochemistry, in second trimester human fetal tissues and in placenta throughout gestation.

Pituitary GH secretion appears largely unnecessary for the attainment of normal birth size in many species, including man. This is believed to be due to an immaturity and/or an absence of GH receptors in many fetal tissues. However, in vitro studies using late first trimester human fetal tissues have demonstrated mitogenic actions of GH on liver and stimulation of insulin biosynthesis in pancreas. To resolve this discrepancy, we have employed immunocytochemistry to identify the presence and distribution of GH receptors in various human fetal tissues. Fetuses of 14-16 weeks gestation were obtained after therapeutic abortion, tissues were fixed, and immunocytochemistry was performed using monoclonal antibodies against purified rat or rabbit GH receptor. The specificity of staining was confirmed by preabsorption of the antibodies with 1) adult rat liver membranes or 2) human fetal liver membranes, both of which possess specific GH-binding sites, or 3) human fetal skeletal muscle membranes, which do not specifically bind GH. Positive staining was seen in a subpopulation of liver parenchymal cells, in the ductal and endocrine tissue of pancreas, in the germinal layer of the epidermis and the deeper dermal layers of skin, and in the tubular epithelium of kidney. No immunopositive staining was seen in skeletal or cardiac muscle, epiphyseal growth plate, lung, intestine, or adrenal. Positive staining was present in the neuronal cell bodies of the cerebral cortex. GH receptor was also detectable as early as 8 weeks gestation in syncytial layers of the placenta and was maintained until term. Results demonstrate the presence of immunoreactive GH receptor/binding protein in some human fetal tissues early in development. In particular, these results would support a role for GH in the growth and function of liver and pancreas.