A rapid, simple determination of plasma cyclic AMP.

Plasma cyclic AMP content was determined without being extracted, using binding protein obtained from rat liver. EDTA was suitable as an anticoagulant for cyclic AMP estimation. Cyclic AMP further added to EDTA plasma was able to be estimated. The estimated values by plasma dilution were almost the same as the expected values. It was thought that the direct assay was useful for determination of plasma cyclic AMP. Isoproterenol (50 microgram/kg, iv) produced an increase of plasma cyclic AMP level accompanied with a decrease of blood pressure and an increase of heart rate in anesthetized dogs. Cyclic AMP level of peripheral venous plasma was 18.6 +/- 1.32 p mole/ml in human (N=25), 21.6 +/- 3.04 P mole/ml in dogs (N=7) and 50.6 +/- 4.59 p mole/ml in rabbit (N=9). Plasma cyclic AMP level of rabbit was higher than those of human and dog.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat.

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D2, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parthyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the "down-regulation" of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Inhibition of apical membrane enzyme activities and protein synthesis by gentamicin in a kidney epithelial cell line LLC-PK1.

The mechanism of a gentamicin-induced decrease in apical membrane enzyme activities was investigated in LLC-PK1 cells. Increasing activities of apical membrane enzymes (alkaline phosphatase, aminopeptidase, and gamma-glutamyltransferase) were markedly suppressed by gentamicin during growth in culture. On the other hand, a lesser effect was observed when the activities of these enzymes were decreasing or relatively constant. Gentamicin treatment decreased the maximal enzyme activities of alkaline phosphatase and aminopeptidase, indicating that the number of active enzyme molecules in the apical membrane was decreased by gentamicin. [3H]Leucine incorporation in LLC-PK1 cells was inhibited by gentamicin in a dose-dependent manner, followed by a reduction of total protein. In addition, a well-known protein synthesis inhibitor, cycloheximide, also decreased the apical enzyme activities. These results suggest that the inhibition of protein synthesis by gentamicin is a possible cause of the decreased activities of apical membrane enzymes in LLC-PK1 cells. The inhibition of protein synthesis may be related to the nephrotoxicity induced by aminoglycoside antibiotics.     

Proteins of the hepatoma tissue culture cell plasma membrane.

The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.     

ATP-stimulated transmitter release and cyclic AMP synthesis in isolated chromaffin granules.

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the beta-gamma position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the alpha-beta position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the beta-gamma linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the alpha-beta position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.     

Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver

We isolated and characterized a subcellular fraction derived from the blood-sinusoidal plasma membrane of hepatocytes enriched in caveolin and containing several of the molecular components described to be present in caveolae isolated from other cell types. A morphological study by electron microscopy revealed that it was composed of caveolae-attached membrane profiles. Immunoelectron microscopy of isolated fraction showed the specific labeling of internal caveolae membranes with anti-caveolin antibody. Finally, one- and two-dimensional electrophoresis and Western blotting were used for the biochemical analysis of this new rat liver plasma membrane fraction. From the biochemical and the morphological characterization, we conclude that the caveolae-enriched plasma membrane fraction is a plasma membrane fraction, which originates from specialized regions of the sinusoidal plasma membrane, enriched in caveolae.     

Rapid assay of cyclic AMP phosphodiesterase and 5'-nucleotidase by means of chromatography on cellulose-nitrate membrane strips

A simple chromatographic procedure with the use of modified cellulose-nitrate membrane strips, 80 x 40 mm, has been worked out for the rapid isotopic assay of cyclic AMP (cAMP) phosphodiesterase (EC 3.1.4.17) and 5'-AMP nucleotidase (EC 3.1.3.5) in crude extracts of various tissues from animals and plants. The assay is based on enzymatic conversion of the product to adenine, a relatively inert compound which, in contrast to cAMP and 5'-AMP, is strongly adsorbed by the cellulose-nitrate membrane. Due to this property rapid separation of adenine from the unconverted substrate (cAMP or 5'-AMP) is possible. Commercial 5'-nucleotidase and easily obtainable crude extract of adenosine nucleosidase from barley leaves are used as coupling enzymes for the phosphodiesterase assay. The assay of phosphodiesterase in 0.5-2 microliter of blood (10(-5) to 4.10(-5) units) has been demonstrated on several examples.     

Discovery of a small-molecule inhibitor for kidney ADP-ribosyl cyclase: Implication for intracellular calcium signal mediated by cyclic ADP-ribose

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclase metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.     

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.     

High-performance membrane chromatography of serum and plasma membrane proteins.

Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.     

Isolation of a metastasizing cancer cell line from an aflatoxin B1-induced rat liver tumor.

An attempt was made to isolate cancer cell lines from liver tumors that had been induced by aflatoxin B1 (AFB1) in rats. A clonal cell line named AFB-1 was isolated from a liver tumor that was histologically diagnosed as hepatocellular carcinoma. When AFB-1 cells were inoculated into the subcutaneous tissue at the dorsal region of syngenic animals, they metastasized from the site of inoculation into the abdominal cavity to form many tumor nodules throughout the serous membrane and metastatic foci in the kidney and pancreas. They also metastasized into the thoracic cavity to form metastatic foci in the lung. This is the first instance where a metastasizing AFB1-induced cancer cell line has been isolated.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat.

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the -EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F-, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adrenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in -EFA livers. When glucagon was used in vitro at 1-1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in -EFA membranes than in +EFA. Total Na+, K+ (Mg2+)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent Km and Vmax-5'-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Elevation of intracellular cyclic adenosine 3',5'-monophosphate levels in macrophages activated with lipopolysaccharide for tumor cell killing.

The macrophage activation for tumor cytotoxicity with lipopolysaccharide (LPS) was remarkably inhibited by adding indomethacin (5 x 10(-6) M) or 10mM LiCl which is known to inhibit adenylate cyclase activity. The tumor cytotoxicity of macrophages inhibited with these agents was recovered by adding dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) (10(-4) or 10(-5) M) and furthermore tumor cell killing activity was augmented as compared with LPS-activated macrophage. Macrophages showed a 5 to 6 times increased intracellular cAMP concentration over the control within 30 min when incubated with LPS. However, the increased intracellular cAMP concentration was decreased by adding LiCl (10 mM). Thus, these findings indicate that there is an important relation between intracellular cAMP concentration and the mechanism of macrophage activation. One can then conclude that at least the initial enhancement of intracellular cAMP was important for tumor cell killing as a signal transmission in macrophage activated by LPS.     

Internalization model using rat liver plasma membrane for receptor-mediated endocytosis of epidermal growth factor.

We have demonstrated the internalization of epidermal growth factor (EGF) using membrane isolated from rat liver. The isolated membrane exhibited a saturation curve of the binding of 125I-EGF. Furthermore, competition between the binding of 125I-EGF and unlabeled EGF to the isolated membrane was observed. These results were similar to those obtained from whole hepatocytes. In order to confirm whether or not the present experimental system using the isolated membrane can be used for the study of receptor-mediated endocytosis, the internalization of pre-bound EGF in the isolated membrane was assessed by two different methods. First, acid-insensitive 125I-EGF time-dependently increased following incubation at 37 degrees C. Secondly, EGF became inaccessible to the exogenous 125I-anti-EGF antibody when under the same condition. These processes were dependent on adenosine triphosphate, but independent of Ca2+ and stimulated by guanosine triphosphate. These results demonstrate that receptor-mediated endocytosis occurred in the isolated membrane.     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

The influence of membrane electrode assembly water content on the performance of a polymer electrolyte membrane fuel cell as investigated by 1H NMR microscopy

The relation between the performance of a self-humidifying H(2)/O(2) polymer electrolyte membrane fuel cell and the amount and distribution of water as observed using (1)H NMR microscopy was investigated. The integrated (1)H NMR image signal intensity (proportional to water content) from the region of the polymer electrolyte membrane between the catalyst layers was found to correlate well with the power output of the fuel cell. Several examples are provided which demonstrate the sensitivity of the (1)H NMR image intensity to the operating conditions of the fuel cell. Changes in the O(2)(g) flow rate cause predictable trends in both the power density and the image intensity. Higher power densities, achieved by decreasing the resistance of the external circuit, were found to increase the water in the PEM. An observed plateau of both the power density and the integrated (1)H NMR image signal intensity from the membrane electrode assembly and subsequent decline of the power density is postulated to result from the accumulation of H(2)O(l) in the gas diffusion layer and cathode flow field. The potential of using (1)H NMR microscopy to obtain the absolute water content of the polymer electrolyte membrane is discussed and several recommendations for future research are provided.     

Binding sites of amyloid beta-peptide in cell plasma membrane and implications for Alzheimer's disease

The binding of amyloid beta peptides (Abeta) to plasma membranes appears to be a promising point of intervention in the events leading to the development of Alzheimer's disease (AD). This binding has been studied as regards the direct toxicity of Abeta on neurons, and the activation of a local inflammation phase involving microglia. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteoglycans and proteins. This review focuses on the membrane proteins that can mediate the interaction between Abeta and the plasma membranes in AD. On neurons, these are APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), integrins, the alpha7nicotinic acetylcholine receptor (alpha7nAChR), the P75 neurotrophin receptor (P75NTR) and the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV). On glial cells, FPRL1 (formyl peptide receptor-like 1), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha(6)beta(1)-integrin and CD47, and heparan sulfate proteoglycans have been reported to bind Abeta. It should be noted that integrins, RAGE (receptor for advanced glycosylation end-products), the Serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind Abeta and are present on neurons and on glial cells. After a presentation of the structure and the function of each of these proteins, the method used to prove their binding to Abeta is described, and the implication of this binding in AD is discussed. Finally, it is underlined that multireceptor complexes containing integrins may be involved in this interaction.     

The band 3 protein of the human red cell membrane: a review.

Band 3 is the predominant polypeptide and the purported mediator of anion transport in the human erythrocyte membrane. Against a background of minor and apparently unrelated polypeptides of similar electrophoretic mobility, and despite apparent heterogeneity in its glycosylation, the bulk of band 3 exhibits uniform and characteristic behavior. This integral glycoprotein appears to exist as a noncovalent dimer of two approximately 93,000-dalton chains which span the membrane asymmetrically. The protein is hydrophobic in its composition and in its behavior in aqueous solution and is best solubilized and purified in detergent. It can be cleaved while membrane-bound into large, topographically defined segments. An integral, outer-surface, 38,000-dalton fragment bears most of the band 3 carbohydrate. A 17,000-dalton, hydrophobic glycopeptide fragment spans the membrane. A approximately 40,000-dalton hydrophilic segment represents the cytoplasmic domain. In vitro, glyceraldehyde 3-P dehydrogenase and aldolase bind reversibly, in a metabolie-sensitive fashion, to this cytoplasmic segment. The cytoplasmic domain also bears the amino terminus of this polypeptide, in contrast to other integral membrane proteins. Recent electron microscopic analysis suggests that the poles of the band 3 molecule can be seen by freeze-etching at the two original membrane surfaces, while freeze-fracture reveals the transmembrane disposition of band 3 dimer particles. There is strong evidence that band 3 mediates 1:1 anion exchange across the membrane through a conformational cycle while remaining fixed and asymmetrical. Its cytoplasmic pole can be variously perturbed and even excised without a significant alteration of transport function. However, digestion of the outer-surface region leads to inhibition of transport, so that both this segment and the membrane-spanning piece (which is selectively labeled by covalent inhibitors of transport) may be presumed to be involved in transport. Genetic polymorphism has been observed in the structure and immunogenicity of the band 3 polypeptide but this feature has not been related to variation in anion transport or other band 3 activities.