Enhancement of intracellular calcium concentration by extracellular ATP and UTP in Madin Darby Canine Kidney cells

Fura2 - fluorescence was utilized to test for the effect of extracellular nucleotides on intracellular calcium concentration of subconfluent Madin-Darby Canine Kidney (MDCK)-cells. Extracellular ATP (10 mumol/l) and UTP (10 mumol/l) lead to rapid (within seconds), sustained, and fully reversible enhancement of intracellular calcium concentration from 138 +/- 9 nmol/l (n = 27), to 1561 +/- 260 nmol/l (n = 10) and 3435 +/- 949 nmol/l (n = 5), respectively. Half maximal effects are observed at some 1 mumol/l. In the absence of extracellular calcium the effect of ATP is transient, pointing to release of intracellular calcium. The sustained effect in the presence of extracellular calcium indicates that the nucleotides in addition recruit calcium from extracellular space.     

Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells

ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources.     

cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain

A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.     

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.     

Defects in protein modification precede developmental defects in l(3)c21RRW630, a temperature-sensitive Drosophila developmental mutant

The temperature-sensitive Drosophila developmental mutation, l(3)c21RRW630 (abbreviated RW630) disturbs oogenesis and has a maternal effect on embryogenesis. At restrictive temperature, RW630 alters post-translational modification of three abundant proteins. To examine the causal relationship between these biochemical defects and the developmental defects in RW630, a series of temperature-shift experiments was performed. It was found that defects in protein modification could be detected in RW630 ovaries after RW630 females had been exposed to restrictive temperature for 1 day. RW630 females treated in this fashion produce embryos which contain a low level of unmodified proteins. Nevertheless, these embryos hatch at a normal rate. Since these ovaries and these embryos are developmentally normal, but do show defects in protein modification, it is unlikely that the RW630 developmental defects cause the biochemical defects in RW630. It is more likely that accumulation of unmodified proteins after extended exposure to restrictive temperature produces the developmental defects in RW630.     

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.     

Molecular mobility and nucleocytoplasmic flux in hepatoma cells

Fluorescence microphotolysis (photobleaching) was used to measure, in single polyethylene glycol-induced polykaryons of hepatoma tissue culture cells, nucleocytoplasmic flux and intracellular mobility for a series of dextrans ranging in molecular mass from 3 to 150 kD and for bovine serum albumin. For the dextrans, the cytoplasmic and the nucleoplasmic translational diffusion coefficients amounted to approximately 9 and approximately 15%, respectively, of the value in dilute buffer. The diffusion coefficients depended inversely on molecular radius, suggesting that diffusion was dominated by viscosity effects. By application of the Stokes-Einstein equation, cytoplasmic and nucleoplasmic viscosities were derived to be 6.6 and 8.1 cP, respectively, at 23 degrees C. Between 10 and 37 degrees C nucleoplasmic diffusion coefficients increased by approximately 45-85%, whereas cytoplasmic diffusion coefficients were virtually independent of temperature. In contrast to that of the dextrans, diffusion of bovine serum albumin was more restricted. In the cytoplasm the diffusion coefficient was approximately 1.5% of the value in dilute buffer; in the nucleus albumin was largely immobile. This indicated that albumin mobility is dominated by association with immobile cellular structures. Nucleocytoplasmic flux of dextrans depended inversely on molecular mass with an exclusion limit between 17 and 41 kD. This agrees with previous measurements on primary hepatocytes (Peters, R., 1984, EMBO [Eur. Mol. Biol. Organ.] J. 3:1831-1836), suggesting that in both cell types the nuclear envelope has properties of a molecular sieve with a functional pore radius of approximately 55 A.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.