Low incidence of heparin-induced skin lesions in orthopedic surgery patients with low-molecular-weight heparins.

BACKGROUND: Heparins are widely prescribed for prevention and therapy of arterial and venous thromboembolic diseases. Heparin-induced skin lesions are the most frequent adverse effect of subcutaneous heparin treatment in non-surgical patients (7.5%-39.8%); no data exist on surgical patients. Commonly, they are due to a delayed-type hypersensitivity reaction (DTH), but may also be a manifestation of life-threatening heparin-induced thrombocytopenia (HIT). Lesions of both entities resemble initially. The risk of HIT is highest among heparin-anticoagulated orthopedic surgery patients. OBJECTIVE: To determine incidence and causes of heparin-induced skin lesions in major orthopedic surgery patients. METHODS: In a prospective cohort study, consecutive patients with subcutaneous low-molecular-weight heparin (LMWH) treatment were examined for cutaneous adverse effects. Further diagnostics (skin biopsy, clinical/laboratory assessment for thrombosis, bleeding, HIT, cross-allergies) were performed. RESULTS: Six of 316 enrolled patients (1.9%; 95% CI: 0.4%-3.4%) developed heparin-induced skin lesions. All were caused by a DTH reaction, and none was due to HIT or other rare heparin-associated skin diseases. Therapeutic use (dosage) of LMWH was identified as only risk factor (odds ratio: 3.1, 95% CI: 1.4-4.9; P = .00141). In addition to DTH, 5 thromboembolic, 4 major bleeding complications but no cases of HIT or cross-allergies were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Orthopedic surgery patients have-unlike non-surgical patients-a low risk for heparin-induced skin lesions during LMWH treatment; all lesions were due to a DTH reaction. The risk for DTH differs considerably between individual patient cohorts. No association with HIT was observed. These data help to tailor anticoagulatory treatment individually and to increase patient safety.

Impact of baseline atrial fibrillation cycle length on acute and long-term outcome of persistent atrial fibrillation ablation.

BACKGROUND: A short baseline atrial fibrillation (AF) cycle length (CL) has been associated with a worse outcome after catheter ablation for AF, whereas the impact of a long baseline AFCL is unknown. We investigated the influence of AFCL on acute and long-term success in a large series of patients undergoing catheter ablation for persistent AF. METHODS: Overall, 177 consecutive patients undergoing catheter ablation of persistent AF using a sequential ablation approach were included in the analysis. AFCL was measured in the left atrial appendage (LAA) at baseline and following each ablation step. The primary endpoint was freedom from any atrial arrhythmia off antiarrhythmic drugs (AAD) with a single ablation procedure after 12 months. RESULTS: Mean AFCL was 164 ± 24 ms. A shorter AFCL was associated with longer AF duration, larger LA diameter, and longer procedure duration. Termination to sinus rhythm (SR) was achieved in 57 (32 %) patients. Baseline AFCL was shorter (161 ± 24 ms) in patients without AF termination compared to patients with AF termination (169 ± 23 m, p = 0.03). The primary endpoint was reached less frequently in patients with a short (<155 ms) AFCL (18 vs. 38.5 %, p = 0.006). Patients with an AFCL between 155 and 200 ms had the best outcome compared to patients with AFCL <155 or ≥200 ms (40 vs. 18 %, p = 0.003). CONCLUSIONS: Patients with a baseline AFCL between 155 and 200 ms have the best outcome after a single ablation procedure for persistent AF compared to patients with an AFCL of <155 or ≥200 ms.

Comparison of the acute pharmacodynamic responses after single doses of ephedrine or sibutramine in healthy, overweight volunteers.

OBJECTIVE: With an increase in the incidence of obesity, tremendous effort has been devoted to the development of weight loss agents and the prospective surrogate markers of both a product's efficacy and safety. The objective of the present study was to compare the pharmacodynamic responses of ephedrine and sibutramine using surrogate markers of weight loss potential and potential adverse events. DESIGN AND SUBJECTS: The study was designed as a 5-way, randomized, double-blinded, placebo-controlled trial with 3 single doses of ephedrine sulfate (0.25, 0.5 and 1 mg x kg(-1)) followed by an open-labeled sibutramine (10 mg) treatment. Healthy, mildly overweight (BMI = 25) subjects were administered the respective treatment and pharmacokinetic and pharmacodynamic measurements (body surface temperature, resting metabolic rate, blood pressure, heart rate, glucose, glycerol, nonesterified fatty acids, triglycerides) were obtained for 8 hours post dose and for an additional 4 measurements during the sibutramine treatment period. RESULTS: Sibutramine treatment significantly increased resting metabolic rate compared to the placebo condition. Ephedrine significantly increased heart rate, systolic blood pressure and glucose but did not significantly affect other measurements. CONCLUSION: Both sibutramine and ephedrine have been shown to have weight loss potential, however, they elicit different metabolic and biochemical responses after a single dose. The nontherapeutic responses from these types of compounds may serve as a screening tool for the development of agents in the treatment of obesity.

HIV protease inhibitors block adipogenesis and increase lipolysis in vitro.

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.

Stimulation of vitamin A(1) acid signaling by the HIV protease inhibitor indinavir.

HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A(1) acid (all-trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5, 5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di-tert-butylchalcone 4'-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.

Drug-antibody conjugates with anti-HIV activity.

Human immunodeficiency virus (HIV)-specific peptide antibody-brefeldin A conjugates and antibody-glaucarubolone conjugates directed to cell surface viral glycoprotein epitopes were prepared and tested for antiviral activity. A selective response was observed both on survival of cell lines permanently infected with lentiviruses and on HIV infectivity. With human peripheral blood mononuclear cells (PBMCs), the conjugate also was effective in reducing virus titers. The effectiveness of an HIV-specific peptide antibody-brefeldin A conjugate was enhanced by combination with 3'-azido-3'-deoxythymidine (AZT) and was effective against AZT-resistant isolates in combination with AZT. The conjugates reduced virus production in MOLT-4 cells and in HIV-1-infected PBMCs without affecting the viability of uninfected cells.

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.

The RXR agonist LG100268 causes hepatomegaly, improves glycaemic control and decreases cardiovascular risk and cachexia in diabetic mice suffering from pancreatic beta-cell dysfunction.

AIMS/HYPOTHESIS: Although retinoid X receptor (RXR) and peroxisome proliferator activated receptor-gamma (PPARgamma) agonists have antidiabetic effects in hyperinsulinaemic animals, little information exists on their effects after pancreatic beta-cell failure. Thus, we examined if RXR and PPARgamma agonists alter distinct metabolic pathways in animals suffering from impaired insulin secretion. METHODS: Adverse side effects and antidiabetic responses were measured in db/db mice treated from 14-16 weeks of age with the RXR agonist, LG100268, and/or the PPARgamma agonists, BRL49653 or GW1929. RESULTS: In animals treated with LG100268 or BRL49653, serum glucose, glycohaemoglobin and the cardiovascular risk factor, fibrinogen, decreased to the same extent. Both of these agonists were equally effective at increasing insulin accumulation in beta cells, although neither agent had an effect on serum insulin concentrations. In contrast, the RXR agonist was less effective than the PPARgamma agonists at lowering serum triglycerides and non-esterified fatty acids and increasing interscapular brown fat and body weight. Further, LG100268 increased serum alkaline phosphatase and liver mass, hepatic fat accumulation, lauric acid hydroxylase activity, catalase-immunostaining and peroxisomal number more than the PPARgamma agonists. Moreover, co-treatment with the RXR and PPARgamma agonists reduced glucose, triglycerides, non-esterified fatty acids and cholesterol more than either agent alone. CONCLUSION/INTERPRETATION: These data suggest 1) RXR and PPARgamma agonists decrease islet degeneration, cardiovascular risk and cachexia during later stages of diabetes, 2) RXR agonists are less effective than PPARgamma agonists at decreasing serum lipids and causing weight gain and 3) RXR agonists have a more pronounced effect on liver metabolism (e.g. peroxisome accumulation and hepatomegaly) than PPARgamma agonists.

Development of infrared imaging to measure thermogenesis in cell culture: thermogenic effects of uncoupling protein-2, troglitazone, and beta-adrenoceptor agonists.

PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.

Effects of troglitazone and metformin on glucose and lipid metabolism: alterations of two distinct molecular pathways.

Troglitazone and metformin are antidiabetic agents that belong to the thiazolidinedione and biguanide classes of drugs, respectively. To evaluate how these drugs influence fuel utilization, we compared their effects on several pathways regulating carbohydrate and lipid metabolism in vitro. Both drugs stimulated glucose transport and utilization in C3H10T1/2 cells, a cell line capable of differentiating into adipocytes when treated with thiazolidinediones. However, we observed that these drugs had a number of different in vitro effects. Unlike metformin, troglitazone stimulated beta3-adrenergic receptor-mediated lipolysis, lipogenesis, and transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Further, by using a mitochondrial-specific fluorescent dye, we found troglitazone to be more effective than metformin at increasing mitochondrial mass. In contrast to troglitazone, metformin was more effective at increasing mitochondrial fatty acid beta-oxidation, peroxisomal fatty acid beta-oxidation, and anaerobic respiration (i.e. lactate production). Additionally, metformin stimulated and troglitazone inhibited both aerobic respiration and basal lipolysis. Insulin enhanced the effects of troglitazone, but not those of metformin, on these cells. Taken together, the data show that troglitazone and metformin affect two distinct metabolic pathways: one that is anabolic (i.e. troglitazone) and the other that is catabolic (i.e. metformin). Further, these observations suggest that the metabolic activity of mitochondria may be lower in cells treated with troglitazone than with metformin.

Thiazolidinediones inhibit alkaline phosphatase activity while increasing expression of uncoupling protein, deiodinase, and increasing mitochondrial mass in C3H10T1/2 cells.

Although there are a number of cell lines committed to differentiate into brown adipocytes, the stem-cell origin of brown fat remains unclear. To address this problem, we explored the effects of various pharmacological agents on differentiation of C3H10T1/2 cells, a pluripotent stem-cell line of mesodermal origin. Histochemical and biochemical analysis revealed that, when these cells were treated with retinoic acid, they expressed the osteoblastic marker alkaline phosphatase. Upon addition of thiazolidinediones and insulin, these cells accumulated lipid and expressed the adipocyte marker aP2, indicating differentiation into adipocytes. Treatment during the growth phase with thiazolidinediones resulted in maximal lipogenesis indicating a need for clonal expansion for efficient adipogenic differentiation. Further analysis revealed that addition of thiazolidinediones to the cells increased (1) the lipolytic response of the cells to beta3-agonists, (2) the expression of uncoupling protein (UCP), (3) the expression of mRNA for type II iodothyronine 5'-deiodinase (5'D-II), and (4) mitochondrial staining. These results suggest the anti-diabetic effects of thiazolidinediones may, in part, involve increased brown adipocyte differentiation. Moreover, this is the first direct evidence indicating that brown adipocytes and osteoblasts may arise from the same stem cell.

Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians.

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109-121, 178-195, 254-271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTP gamma S, and GDP beta S but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109-121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109-121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis.

Is the drug-responsive NADH oxidase of the cancer cell plasma membrane a molecular target for adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC50 for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.

Identification of antitumor sulfonylurea binding proteins of HeLa plasma membranes.

Plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of about 25 nM). The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance protein of the total plasma membrane proteins. The binding proteins appeared to contain one or more thiols in the binding site as high affinity binding of [3H]LY181984 was reduced by treatment with the covalent thiol blocking reagent, N-ethylmaleimide (NEM), or by oxidation with dilute hydrogen peroxide but was protected by glutathione or dithiothreitol. Elimination of binding of [3H]LY181984 by NEM was prevented by excess unlabeled LY181984 (an active sulfonylurea) but less so by excess LY181985 (an inactive sulfonylurea). The binding proteins were specifically labeled with thiol reagents following reaction of unprotected thiols with unlabeled thiol reagents. Binding proteins at ca. 34 kDa were labeled. Plasma membrane proteins after solubilization with SDS under strongly reducing conditions still bound sulfonylurea. [3H]LY181984 binding to plasma membrane proteins resolved on SDS-PAGE correlated as well with proteins in the 30-40 kDa range.

A 55 kDa protein of transitional endoplasmic reticulum from rat liver binds retinol.

A transitional endoplasmic reticulum fraction from rat liver that responds to retinol by increased formation of 50-70 nm diameter transition vesicles bound retinol. Separation on SDS-PAGE with analysis by fluorography indicated the dominant binding component at 55 kDa. Binding was found with transitional vesicles formed from the transitional endoplasmic reticulum, and the endoplasmic reticulum fractions from which vesicles are derived but not with Golgi apparatus, plasma membranes or cytosol.

Inhibition by brefeldin A of NADH oxidation activity of rat liver Golgi apparatus accelerated by GDP.

Reduced pyridine nucleotide has been reported to enhance cell-free transfer of membrane material from a radiolabeled Golgi apparatus donor fraction from rat liver to an acceptor fraction consisting of inside-out plasma vesicles immobilized on nitrocellulose [(1992) Biochim. Biophys. Acta 1107, 131]. As part of a continuing effort to identify NADH-requiring enzymes in the Golgi apparatus which may be important to membrane trafficking, highly purified fractions of Golgi apparatus from rat liver were tested for their ability to oxidize NADH and the inhibition of the oxidation of NADH by brefeldin A. The isolated Golgi apparatus fractions were found to oxidize NADH with a specific activity comparable to that of the plasma membrane of rat liver. The activity was inhibited by brefeldin A and this inhibition was augmented by GDP. At near optimal concentrations of 7 microM brefeldin A and 1 microM GDP, the activity was > 90% inhibited. Brefeldin A inhibition of NADH oxidation by the Golgi apparatus was time-dependent and GDP appeared to accelerate the inhibition by brefeldin A.

Modulation of guanine triphosphate nucleotide binding to p21ras immunoprecipitates of rat liver plasma membranes by agents affecting redox state.

GTP-gamma-[35S] and GTP-gamma-[32P] or GTP-alpha-[32P] bound to plasma membranes of rat liver was immunoprecipitated using anti p21V-H-ras. Binding was enhanced approximately 2-fold by incubation with an exogenous electron acceptor, potassium ferricyanide (but not with potassium ferrocyanide), or oxidized ubiquinone10 and was inhibited or unaffected by incubation with reduced pyridine nucleotides (NADH or NADPH) or reduced ubiquinone10. The results suggest a mechanism of guanine nucleotide exchange that is responsive to oxidation-reduction.

Temperature- and acceptor-specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to the cis Golgi apparatus.

The temperature dependence and specificity of transfer of membrane constituents from donor transitional endoplasmic reticulum to the cis Golgi apparatus were investigated using a cell-free system from rat liver. The radiolabelled transitional endoplasmic reticulum donors were prepared from slices of rat liver prelabelled with [14C]leucine. The acceptor Golgi apparatus elements were unlabelled and immobilized on nitrocellulose. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from the cis, medial and trans portions of the stack respectively, efficient specific transfer was observed only to cis elements. Trans elements were devoid of specific acceptor capacity. Similarly, when transfer was determined as a function of temperature, a transition was observed in transfer activity between 12 degrees C and 18 degrees C similar to that seen in vivo for formation of the so-called 16 degrees C cis Golgi-located membrane compartment. Transfer at temperatures below 16 degrees C and transfer to trans Golgi apparatus compartments at temperatures either above or below 16 degrees C was similar and unspecific. The unspecific transfer at low temperature was pH independent, whereas specific transfer was greatest at the physiological pH of 7, and was reduced to 10% and 18% of that occurring at pH 8 and pH 5.5 respectively. These findings show that the cell-free system derived from rat liver exhibits a high degree of fidelity to transfer in vivo, an efficiency approaching that observed in vivo, and a nearly absolute acceptor specificity for cis Golgi apparatus. The acceptor-, temperature- and pH-specificity of the cell-free transfer, as well as the saturation kinetics exhibited with respect to acceptor Golgi apparatus, support the concept of transition-vesicle-specific docking sites of finite number associated with cis Golgi apparatus cisternae.

NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver.

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.