Leon A. Heppel

Extracellular ATP shows synergistic enhancement of DNA synthesis when combined with agents that are active in wound healing or as neurotransmitters

The polypeptides PDGF, TGF alpha, and EGF have previously been shown by others to stimulate proliferation of fibroblasts and keratinocytes in the process of wound healing. Here we demonstrate that extracellular ATP, ADP or AMPPNP caused synergistic enhancement of DNA synthesis in 3T6 mouse fibroblasts and BALB/MK keratinocytes when combined with any of the above polypeptides. TGF beta showed synergistic stimulation with ATP in fibroblasts but it inhibited keratinocytes. ATP acted as a mitogen for NIE-115 neuroblastoma cultures. In 3T6 cells, ATP stimulated thymidine incorporation in combination with carbachol or norepinephrine. The effect of carbachol was sensitive to atropine. We suggest that extracellular ATP and ADP may play a physiological role in wound healing and as a mitogenic neurotransmitter in the nervous system.

Permeabilization of Transformed Cells in Culture by External ATP

procedures include the use of ionophores, manipulation of osmotic conditions, treatment with ouabain or with agents that reduce the level of intracellular ATP, and also the use of inhibitors of Ca ++calmodulin systems.

Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization.

The stimulation of calcium efflux from Swiss 3T6 mouse fibroblasts by extracellular ATP was studied. It was found that the cells could be desensitized to ATP by a previous exposure to the nucleotide, lending support to the theory that this is a receptor mediated process. Another ATP-receptor mediated process in Swiss 3T6 cells, that is also subject to desensitization, causes the permeabilization of the plasma membrane to nucleotides and other normally impermeant compounds [Gonzalez et al., J. Cell. Physiol. 139:109 (1989)]. Here we demonstrate that selective desensitization of the ATP-dependent calcium mobilization pathway can be achieved without affecting ATP-induced permeabilization. Data are presented in support of the existence of multiple ATP-receptors (purinoceptors) in Swiss 3T6 cells.

Purification of the membrane-bound and pyridine nucleotide-independent L-glycerol 3-phosphate dehydrogenase from Escherichia, coli

Abstract The membrane-bound L-glycerol 3-phosphate dehydrogenase has been solubilized from Escherichia , coli by extraction with sodium deoxycholate dissolved in 0.25 M NaCl. Fractionation by DEAE-cellulose and phosphocellulose chromatography in the presence of 0.2% Brij 58 yielded a preparation that was practically homogeneous. The purified enzyme had a Km of 0.8 mM for DL-glycerol 3-phosphate compared with 23 mM for the membrane preparations. The enzyme comprises about 1% of membrane protein in a mutant constitutive for L-glycerol 3-phosphate dehydrogenase. Its flavin coenzyme is FAD.

The effect of oncomodulin on cAMP phosphodiesterase activity

Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43, 5390-5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: Studies on individual cells using quantitative digital video fluorescence microscopy

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.

THE MECHANISM OF ACTION OF POLYNUCLEOTIDE PHOSPHORYLASE

The purpose of this paper is to review certain aspects of the mechanism of action of polynucleotide phosphorylase and to present, rather briefly, some recent findings. The discussion will be concerned with studies carried out by S. Ochoa and his associates a t New York University, New York, N. Y., and with work done a t the National Institutes of Health. Reference will also be made to some recent work carried out in Bethesda by Grunberg-Manago. Some of the material to be presented has already been published, but a review may be profitable at this time. Certain of the unsolved problems that face investigators in this field also will be discussed. Polynucleotide phosphorylase was discovered by Grunberg-Manago and Ochoa in extracts of Azotobacter agile.’r2 Studies of the nature of nucleotide incorporation into nucleic acid in Escherichia coli led to a recognition of the same reaction by Littauer and Rornberg.3, 4 , Beers6 has made extensive studies of the enzyme from Micrococcus lysodeikticus, and some of this work will be presented in another paper in this symposium. Olmsted’ has also reported studies dealing with polynucleotide phosphorylase from M . lysodeikticus. The reaction catalyzed by the enzyme may be formulated as follows:

Extracellular ATP stimulates increases in Na+K+ pump activity, intracellular pH and uridine uptake in cultures of mammalian cells

Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L. A. (1989) Proc. Natl. Acad. Sci. USA 86, 7904-7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and Na+/K+ pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.

STUDIES ON BINDING PROTEINS, PERIPLASMIC ENZYMES AND ACTIVE TRANSPORT IN ESCHERICHIA COLI

Abstract Binding proteins, which are considered to be involved in active transport, are selectively released from E. coli and related bacteria by osmotic shock. Certain degradative enzymes are also set free. A mutant which forms surface membrane vesicles in stationary phase has recently been studied. Several of the enzymes released by osmotic shock were examined and found to be contained in these vesicles. Amino acid transport systems differ with respect to the effect of osmotic shock. In some cases transport is reduced by shock and a binding protein with the same specificity is released into the shock fluid; this is true for the glut amine uptake system. In other cases, amino acid transport is not lost by osmotic shock and no binding protein with the corresponding specificity is set free. The lysine-specific uptake system is an example and it is present in vesicles made by Kabacks procedure. Lysine-specific binding activity has been eluted from vesicles with buffers of low ionic strength. Evidence indicates that, in both whole cells and vesicles, α-glycerophosphate must be transported across the membrane in order to be oxidized and provide energy for transport of amino acids.

Evidence for a role of G protein βγ subunits in the enhancement of cAMP accumulation and DNA synthesis by adenosine in human cells

The expression of both A1‐ and A2a‐adenosine receptors occurs in human foreskin and lung fibroblasts (Ahmed et al., 1995, Biochem. Biophys. Res. Commun. 208:871–878). Studies with highly specific A1‐ and A2a‐adenosine receptor agonists provide indirect evidence that binding of adenosine activates Gs and Gi, after which Gsα interacts with βγ subunits released from Gi. The interaction of Gsα with βγ augments cyclic adenosine monophosphate (cAMP) accumulation, more than does Gsα alone. In the present study, we have provided direct evidence for a role of the βγ complex in the augmentation of cAMP accumulation by using a recombinant His6 fusion protein containing the carboxyl third of βARK1. This portion of βARK1 contains Gβγ binding sequences and acts as a specific βγ scavenger (Koch et al.,1994, Proc. Natl. Acad. Sci. USA 1:12706–12710). In permeabilized fibroblasts, the His6 fusion protein inhibited the augmentation of cAMP accumulation resulting from adenosine binding to both A1 and A2a receptors. In addition, the specific Gβγ scavenger inhibited the further rise in cellular cAMP levels caused by pretreating cells with pertussis toxin before incubation with adenosine. Finally, we observed that specific A1‐adenosine receptor agonists augmented the cAMP accumulation stimulated by A2a‐receptor agonists, and this cAMP augmentation was also suppressed by the Gβγ scavenger. Similar results were obtained when the cells were treated with extracellular ATP and lysophosphatidic acid (LPA) to stimulate Gs and release Gβγ subunits, respectively. J. Cell. Physiol. 170:263–271, 1997.