ANNUAL RIPORT OF THE LABORATORY OF BIOCUSMICALGENETICS NATIONAL HEART AND LUNG INSTITUTE July 1, 1975 through June 30, 1976 Fusion of clonal neuroblastoma cells with rat glioma cells yielded clonal hybrid cell lines which synthesize, store and excrete acetylcholine; proper- ties which are not expressed by the parental cell lines. Cells from one hybrid line were found to form synapses with cultured striated muscle cells. Synapses between hybrid cells ar&muscle cells closely resemble the synapses between normal motor neurons an&striated muscle before they are fully developed. Underappropriatie con&t-. hybrid cells establish synaptic connections with virtually every muscle.cell tested; thus,.synaptic connections are formed in abundanceL.:.Martted differences were observed in the efficiency of transmission across diffekeat syrt#e&.:~Axomal activities~whkch were foyad to be regulated include--choline &etyltramsferase,,. acetylcholinesterase, Na action potential ionophos&s@ecific ac&itiea,.and.thc rate.oE.choline.transport into cells. :Eight:'species of r&eptors~h&e~heen found thms far-with the hybrid cell ,Ilne-uhicbfusms syn~&'...Reeeptor mediated`shifta in c&P levels, cGMP levels and.?naskaeipotmt&Euti bee&identified&+characterized.. Thus,the foundation has been laid'.for studies.on the effects-of receptor-mediated reac-- tions~on.synaptic transmission, IR additiort;~re than 100 cell lines which 'synthesf&acetylcholinehave been obtained and are being studied to determine whether-e cell lines. are defective with respect to synapse formation. Much remains-to he done but it seems clear that the experimental approach and model 'systems which have been-established afford extraordinary opportunities to ex- plore synapse properties-and correlate biochemical events with developmental and electrophysiological phenomena. Synaptogenesis by normal neurons also was studied. Neurons dissociated from chick embryo retina and maintaine !? in vitro were found to reaggregate -- and form in vitro approximately 1 x 10 Three -.- synapses per mg of protein. types of synapses and several subtypes were identified which closely resemble those of the intact retina, Studies with this system are described in other sections of this report. A histochemical technique for detecting and.localizing nicotinic acetyl- choline receptors was devised previously which depends upon the formation of a complex between peroxidase.coupled to an antibody for a-bungarotoxin and the nicotinic acetylcholine receptor, Using this method, clusters of nicotinic acetylchctline-receptors on cultured muscle cells were shown to contain at least 7 times the concentration of receptors found in other.membrane regions. Recep- tor clusters are not characteristically associated with folds in the plasma nembrane, these receptors. Exp &ate receptors to morphine for 12 t ctivity w,, S Cells have normal CAP@ levels and appear tolerant to morphine because the increase in adenylate cyclase activity is approximately equal to the inhibitio~~-e&Z&ty. we. However, the cells then are dependent upon morphine to maintain normal CAMP levels. Withdrawal of morphine, or displacement of the narcotic from the opiate receptor by the antagonist, naloxone, reverses the inhibition and results in the synthesis of abnormally high levels of CAMP. Thus, dual regulation of adenyl that the endogenous opiate peptides and narcotics act as pleiotropic regulators of other species of receptors which are coupled to the activation of adenylate cyclase. By . . 'this mechanism opiatesalter the perception of neurons to incoming messages. A cell line with minic inhibitory acetylcholine receptors, and 'another line. with muscarinic excitatory receptors were found. The number of ,receptors, the receptor-afffnities for cholinergic 1igancJs and other receptor properties were determined by measuring the binding of [ Hf-quinuclidinyl benzilate to receptors,: ceptor complex is 6 x IO- Tpw e apparent dissociation constant of the ligand-re- Activation of muscarinic acetylcholine recep- tors results in a tramsient &crease in cGMP;and a profound, long-lived inhibition of adenylatc cyclase activity. Exposure of cells to acetylcholine or carbachol for 24 hours marrcedly decreases the number of acetylcholine receptors and increases adenylate cyclase activity 50 to 500%. Removal of carbachol results in the.return of adenylate cyclase activity and acetylcho- line receptor levels to normal values after approximately 3 and 24 hours, respectively. Similarly, exposure of cells with a-receptors to norepinephrine inhibits adenylate cyclase activity and elicits a delayed, compensatory in- crease in adenylate cyclase activity. These results show that prolonged acti- vation of a-receptors, muscarinic acetylcholine receptors, or opiate receptors results in cell tolerance to and dependence upon norepinephrine, acetylcholine, or opiates, respectively. Withdrawal of the receptor activator elevates intra- cellular CAMP levels and shifts cells to a supersensitive state with respect to other species of receptors which activate adenylate cyclase. Desensitization of muscarinic receptors decreases the affinity of the receptor for agonists by a process which exhibits negative cooperativity; whereas interactions between the receptor and antagonists are not cooperative. Both muscarinic excitatory and inhibitory acetylcholine receptors were solu- bilized and the properties of membrane-bound and soluble receptors were com- pared. Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors, Both muscarinic and nicotinic acetylcho- line receptors are synthesized before synapses appear in the retina; however, during development, nicotinic acetylcholine receptors become associated pre- dominantly with neurites in the synaptic layers of the retina. The properties of muscarinic acetylcholine receptors were determined at different develop- mental ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma cells. 2 Evidence for a new type of PGEl receptor coupled to cGMP accumulation was obtained. Cell lines with PGEl receptors coupled only to CAMP were found as well as cell accumulation, lines with 2 species of PGEl receptors, one coupled to CAMP the other to cGW accumulation. also desensitize at different rates. The 2 species of PGEl receptors These results show that the coupling of PGE to increases in CAMP and cGMP levels are clonally inherited properties whit can be expressed independently. Al Two pathways for y-aminobutyric acid synthesis were found in chick embryo retina.' The first pathway depends upon the conversion of putrescine to orni- thine, catalyzed by ornithine decarboxylase and the subsequent conversion of ornithine~ to y-aminobutyric acid. The second route of synthesis is dependent upon the conversion of .glutamic acid to y-aminobutyric acid, catalyzed by glutanric acid decarboxylase. Elevation of CAMP levels in neuroblastoma cells. was shown to induce ornithine decarboxylase activity. In the developing embryo, neurotransmitters which affect CAMP levels may regulate ornithine decarboxylase activity and thereby control the rate of GABA synthesis from ornithine. Previous resultsled to the conclusions that (l)+veratridine, batracho- toxin, and aconitine activate the action potential Na ionophore by interaction with a single class of sites; (2) scorpion venom activates the ionophore by interaction with a different class of sites; (3) two species of toxin bound to separate sites are allosterically coupled and interact in a cooperative manner; and (4) tetrodotoxin and saxitoxin act at a 3rd site which is involved in ion transport. A toxin which activates the action potential Na* ionophore has been puri- fied from scorpion venom. The toxin binds to a single class of sites and acts cooperatively with each of the three alkaloids.' Depolarization of cells causes a 30-fold increase in the apparent dissociation constant. The results svggest that the scorpion toxin binds to a voltage sensitive component of the Na ;;~;;~y;i,'p;nafs5 cooperatively in regulating ion transport activity. Binding I-labelled derivative of scorpion toxin showed that the concentration of toxin binding sites is approximately 3 to 6 fmole per mg protein. Clonal skeletal muscle myoblasts have substantial action potential Na+ ionophore activity. A small increase in activity accompanies cell fusion. The activity in both myoblasts and myotubes is relatively insensitive to inhi- bition by saxitoxin and tetrodotoxin and thus resembles denervated rat striated muscle which has been shown to be relatively insensitive to these toxins. Chronic electrical stimulation of muscle cells in vitro does not increase -- tetrodotoxin sensitivity. At least 3 ionophores are qvolved in the action potential in adult heart: a rapidly activated axon-like Na of the action potentia$, iowpho$e responsible for the rising phase a slower Ca /Na ionophore responsible for the plateau phase, and a K ionophore responsible fqr the repolarization phase. spe ;;;d;;Ew2;4%,N c ific inhibitors of the fast Na ionophore (tetrodotoxin) and a ionophore (D-600) show that the role of these two types of ionophore in beating changes during development of the embryonic chick heart. 3 In early embryonic hearts, the fast Na' ionophore is .present but is not required for beating. culture in vitro, During development in ovo or in monolayer or aggr$gate changes in the requirement for activity of the fast Na ioqophse in beating Na /Ca are accompanied by change? infihe sensitivity of the slow ionophore to D-600. When the slow Na /Ca fonophore is able to maintain beating without participation of the fast Na+ ionophore, its sensitivity to D-600 is $igh+yhereas when the fast Na ionophore is required for beating, the slow Na /Ca ionophore is relatively insensitive to D-600. Transitions between these two states can be induced in 2 hours in vitro by -- inhibition of beating. Thus, the activity of these ionophores is regulated during development by a process dependent on the rhythmic activity of the cells. The developmental changes in action potential ionophores of embryonic hearts occur between days 4 and 7 in ovo. During this time, vagal innervation -- of the heart takes place. Consequently we have studied the muscarinic acetyl- 'choline receptors in embryonic hearts using both physiologic and ligand binding methods to determine whether changes in their properties are temporally correlated with changes in the action potential ionophores. In early embryonic hearts, muscarinic agents are ineffective in inhibiting beating. Between days 5 and 7, sensitivity to inhibition by muscarinic agents increases to,the adult level. QNB binds specifically to muscarinic acetylcholine receptors in embry- onic and adult heart as assessed by competition studies with muscarinic and nicotinic agents. Receptors, as detected by QNB binding, are present in unre- sponsive early embryonic hearts and the number per mg heart protein does not increase dramatically during development. Receptor desensitization is accom- panied by a small change (3 fold3increase) in the KD for muscarinic agonists as measured by competition with H QNB. This change in KD occurs only in hearts that are responsive to muscarinic agents and thus may be associated with the physiologic action of the receptor. Competition curves for agonists suggest the involvement of negative cooperativity in activation of the receptor, These results suggest that muscarinic acetylcholine receptors, like action potential ionophores, are present in early embryonic hearts in modified "precursor" or "inactive" forms and undergo activation during development. Studies on the mechanism of catabolite repression in E. coli show that -- glucose inhibits adenylate cyclase.activity reversibly in cells treated with toluene, and that phosphate is required for high adenylate cyclase activity and for glucose dependent inhibition of enzyme activity. Other sugars also inhibit adenylate cyclase provided that transport systems for the sugars are induced. Mutant strains of E. coli defective in phosphoenolpyruvate:sugar -- phosphotransferase system components were examined. Cyclic AMP levels were normal in an HPr mutant, but were markedly depressed in a leaky Enzyme I mutant. Adenylate cyclase'activity was low in the Enzyme I mutant whereas the HPr mutant had normal enzyme activity. The Enzyme I mutant under starva- tion conditions exhibits high adenylate cyclase activity and the adenylate cyclase of this mutant is unusually sensitive to variations in carbon source. The addition of phosphoenolpyruvate leads to a substantial increase in adenylate cyclase activity in permeabilized cell preparations of the Enzyme I mutant. These results suggest that Enzyme I is involved in the regulation of adenylate cyclase activity. Studies are in progress to test the hypothesis that Enzyme I interacts with adenylate cyclase and that the PEP-dependent phosphorylation of Enzyme I is responsible for activation of adenylate cyclase. 4 Levels of cCMP were compared with CA!.? levels in E. coli grown under -- different conditions. The results show that cCMP and CAMP concentrations are inversely coupled in E. coli and that the regulation of cCMP levels can be -- uncoupled from that of CAMP. Since specific species of tRNA are involved in amino acid mediated repres- sion and end-product inhibition, the effect of amino acid deprivation upon tRNA of relaxed and stringent strains of E. coliwas studied. In relaxed -- control E. coli, leucine starvation results in the formation of new isoaccep- tor species of leucine, histidine, arginine, valine, and alanine-specific tRNA and quantitative changes in the concentration of some other isoacceptors. Experiments with stringent strains or the use of uracil starvation or rifampicin addition provided evidence for the de novo synthesis of new species of -- leucine-tRNA. The new species of leucine-tRNA is not formed by aggregation of tRNA or nuclease catalyzed hydrolysis, and it is not grossly deficient with respect to methylation. Since there is some evidence from the recent work of others that tRNA formed under conditions of amino acid starvation is deficient in the minor bases 5,Gdihydrouridine and 4-thiouridine, we think that the biochemical explanation for the accumulation of new tKNA species under condi- tions of amino-acid starvation may be that the enzymes responsible for these tRNA modifications are unstable and require continued protein synthesis to maintain their levels of activity. Further information was ob-tained on the mechanism of arginyl-tRNA synthe- tase which does not catalyze an amino acid dependent ATP-PPi exchange in the absence of added tRNA. A new purification procedure was devised which yields homogeneous enzyme in approximately 10% yield. Pulse labelling experiments indicate that no enzyme bound arginyl-adenylate is formed in the absence of added tFWA. Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNAarg. These data further validate our earlier suggestion that the mechanism of this reaction is probably concerted. A series of additional studies carried out with the purified preparation of arginyl-tRNA synthetase from E. coli indicate that metals may have two func- -- tional roles in the catalytic mechanism. Complete metal activation is observed when MgCl MnCl CoCl or FeCl is present at a concentration (5 mM) in ex- cess of tg: totaf'ATP c&entrat& (2 mM). activity is not observed unless a small amount (0.1 mM? of MgCl When CaCl is substituted for MgC12, , MnCl FeCl or ZnCl is added. CoC12, 3 ii On the basis of these experiments, we2visual?Le a mode in whit the enzyme possesses a site for free metal which, when filled, lowers the K for the three substances (arginine, tKNAarg, and metal'ATP) and increases thg Vmax of the reaction. 5