Effect of W7 on Ca2+ uptake and Ca2+-ATPase activities of the endoplasmic reticulum of rat liver.

In an initial attempt to use calmodulin antagonists as probes to study the role of calmodulin in the modulation of Ca2+ uptake activity in the endoplasmic reticulum of rat liver, we noticed that W7 had a differential effect on the Ca2+ uptake and Ca2+-ATPase activities. To test the specificity of this effect and explore the underlying mechanism, we examined the effects of W7 on Ca2+ accumulation and release by endoplasmic reticulum in both permeabilized hepatocytes and a subcellular membrane fraction (microsomes) enriched in endoplasmic reticulum. W7 reduced the steady-state Ca2+ accumulation in both preparations in a dose-dependent fashion but the half-maximal inhibitory concentrations were different for Ca2+ accumulation (90 microM) and Ca2+-ATPase activity (500 microM). Kinetic analysis indicated that the inhibition of both Ca2+ uptake and Ca2+-ATPase activity by W7 was noncompetitive with respect to Ca2+ and ATP. Addition of W7 did not enhance the rate of Ca2+ efflux from microsomes after Ca2+ influx had been terminated. The effect of W7 was apparently not related to its calmodulin antagonist properties as the phenomenon could not be demonstrated with the other more specific calmodulin antagonists, calmidazolium or compound 48/80. A similar observation with W7 has also been reported with the endoplasmic reticulum of pancreatic islets (B. A. Wolf, J. R. Colca, and M. L. McDaniel (1986) Biochem. Biophys. Res. Commun. 141, 418-425). We concluded that the effects of W7 on microsomal Ca2+ handling were not the result of increased membrane permeability to Ca2+ but rather were due to dissociation of Ca2+ uptake from Ca2+-ATPase activity.

Chelation of intracellular calcium blocks insulin action in the adipocyte.

The hypothesis that intracellular Ca2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport (IC50, 26 microM quin-2 AM) without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl2 and the Ca2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation (IC50, 11 microM quin-2 AM) and the ability of insulin to inhibit cAMP-stimulated lipolysis (IC50, 78 microM quin-2 AM), without affecting their basal activities. Incubation of cells with 100 microM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125I-labeled insulin to adipocytes. These findings suggest that intracellular Ca2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca2+-dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin.

Criteria for evaluating nonquantitative assays: application to serum choriogonadotropin.

We present guidelines for assessing the performance of nonquantitative (qualitative) assay methods. Criteria to be evaluated include analytical sensitivity, imprecision near limits of detection, analytical specificity, accuracy over a wide range of analyte concentrations, potential interferents, and technical ease of performance. A protocol was developed to evaluate several nonquantitative assay kits for detection of human choriogonadotropin (hCG) in serum. These include Tandem Icon HCG (Hybritech), Quest Pregnancy Test (Quidel), Concep-7 beta hCG (Leeco), and Beta Quik V (Pacific Biotech). Quantitative measurement of beta-hCG by RIA (Immophase beta hCG, Corning Medical) was used as the reference method. Results of this evaluation are discussed. The guidelines established and utilized in this report are adaptable to the evaluation of assay kits that measure other analytes by qualitative techniques.

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

A calmodulin dependent Ca2+-activated K+ channel in the adipocyte plasma membrane.

Increased membrane permeability (conductance) that is specific for K+ and directly activated by Ca2+ ions, has been identified in isolated adipocyte plasma membranes using the K+ analogue, 86Rb+. Activation of these K+ conductance pathways (channels) by free Ca2+ was concentration dependent with a half-maximal effect occurring at 32 +/- 4 nM free Ca2+ (n = 7). Addition of calmodulin further enhanced the Ca2+ activating effect on 86Rb+ uptake (K+ channel activity). Ca2+-dependent 86Rb+ uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K+ channels that are activated by Ca2+ and amplified by calmodulin.

Sulfation of chondroitin oligosaccharides in vitro. Analysis of sulfation ratios.

Microsomal preparations from cultured chick embryo chondrocytes were incubated with 3'-phosphoadenosine 5'-phosphosulfate and oligosaccharides prepared from chondroitin. Rates of 4- and 6-sulfation were measured at pH 6 and 8 in the presence of MnCl2 and Brij 58. Ratios of the overall 6-sulfation to 4-sulfation rates ranged from 40-200 at pH 8 and from 6-35 at pH 6, depending upon the composition of the assay mixture. When saturating concentrations of 3'-phosphoadenosine 5'-phosphosulfate and the oligosaccharide acceptors were used, the resulting products were mixtures of monosulfated oligosaccharides. The compositions of the mixtures formed from oligosaccharides with degrees of polymerization from 4-12 at pH 6 and 8 were analyzed. Sulfate substituents were found at all N-acetyl-D-galactosamine (GalNAc) residues in the acceptors but were not evenly distributed along the oligosaccharide chains. For oligosaccharides with nonreducing terminal D-glucuronic acid (GlcUA) residues, sulfation at the nonreducing terminal GlcUA----GalNAc occurred exclusively at the C6 of the GalNAc residue. However, for oligosaccharides with nonreducing terminal GalNAc residues the rate of 6-sulfation of the nonreducing terminal GalNAc was markedly reduced and was similar to the rate of 4-sulfation at the same position. The rates of sulfation at the reducing ends of the oligosaccharides were relatively high for the shorter oligosaccharide acceptors but decreased with increasing length of the acceptor, suggesting that the sulfotransferases recognized primarily the GalNAc residues in the nonreducing terminal regions.

Vanadyl and vanadate inhibit Ca2+ transport systems of the adipocyte plasma membrane and endoplasmic reticulum.

Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.

Preparation and high-performance liquid chromatography of 3'-phosphoadenosine-5'-phospho[35S]sulfate with a predetermined specific activity.

3'-Phosphoadenosine-5'-phospho[35S]sulfate (PAP35S) was prepared by incubating ATP and carrier-free H2(35)SO4 with a 100,000g supernatant fraction prepared from chick embryo chondrocytes. The product was partially purified by paper electrophoresis and mixed with unlabeled PAPS to give a solution of PAP35S with a specific activity and a concentration approximating those required for the desired metabolic studies. The product was analyzed by high-performance liquid chromatography on an anion-exchange column to determine the proportion of the 35SO4 cpm and A260 material found in the PAPS and other contaminating nucleotides. The PAP35S was purified further by preparative high-performance liquid chromatography. The exact specific activity of the PAP35S was then determined by using this PAP35S preparation as the SO4 donor in a sulfotransferase reaction using a microsomal preparation from the chick embryo chondrocytes as the enzyme and an 3H-labeled oligosaccharide as the SO4 acceptor. The sulfated oligosaccharide was then isolated and the number of 3H and 35SO4 counts per minute in this product were used to calculate the specific activity of the donor. The features of this generally useful approach for preparing PAP35S of any desired specific activity and concentration are discussed.