Draft of "Annual Report of the Laboratory of Biochemical Genetics, October 1, 1975 - September 30, 1976"
These drafts and project reports are from a highly prolific period in the Laboratory of Biochemical Genetics. Major research
projects discussed include: 1) regulation of adenylate cyclase; 2) acetylcholine receptors and nervous system development;
3) the development of chick embryo retina; and 4) morphine receptors. The major findings, significance to biomedical research,
and publications are included for each.
It appears as though page five is misnumbered.
Item is a photocopy.
Number of Image Pages:
11 (814,867 Bytes)
1976-06-14 (June 14, 1976)
Nirenberg, Marshall W.
National Heart and Lung Institute. Laboratory of Biochemical Genetics
This item is in the public domain. It may be used without permission.
From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1975 - September 30, 1976 (September 1976)
Box Number: 13
Folder Number: 35
Fusion of clonal neuroblastoma cells with rat glioma cells yielded clonal hybrid cell lines which synthesize, store and excrete
acetylcholine; properties 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 normal hybrid cells and muscle cells closely resemble the
synapses between motor neurons and striated muscle before they are fully developed. Under appropriate conditions, hybrid
cells establish synaptic connections with virtually every muscle cell tested; thus, synaptic connections are formed in abundance.
Marked differences were observed in the efficiency of transmission across different synapses. Axonal activities which were
found to be regulated include choline acetyltransferase, acetylcholinesterasa, Na+ action potential ionophore specific activities,
and the rate of choline transport into cells. In muscle ce11s, the distribution of nicotinic acetylcholine receptors is regulated.
Eight species of receptors have been found thus far with the hybrid cell line which forms synapses. Receptor mediated shifts
in cAMP levels, cGMP levels and membrane potentials have been identified and characterized. Thus the foundation has been
laid for studies on the effects of receptor mediated reactions on synaptic transmission. In addition, more than 100 cell
lines which synthesize acetylcholine have been obtained. Preliminary results suggest that some cell lines may be defective
with respect to synapse formation.
Although these studies are in their infancy, it seems clear that the experimental approach and model systems which have been
established afford extraordinary opportunities to explore synapse properties and to correlate biochemical events with developmental
and electrophysiological phenomena.
Synaptogenesis by normal neurons also was. Neurons dissociated from chick embryo retina and maintained in vitro reaggregate
and were also shown to form in vitro approximately 1 x 10^9 synapses per mg of protein. Three 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 preliminary histochemical technique for detecting and localizing nicotinic acetylcholine receptors was devised which depends
upon the formation of a complex between peroxidase coupled to an antibody for alpha-bungarotoxin and the nicotinic acetylcholine
receptor. Using this method, clusters of nicotinic acetylcholine receptors on cultured muscle cells were shown to contain
at least 7 times the concentration of receptors found in other membrane regions. Receptor clusters were not characteristically
associated with folds in the plasma membrane.
The hybrid cells which form synapses possess abundant morphine receptors. Morphine and other narcotics were shown to be potent
inhibitors of adenylate cyclase in cells which possess opiate receptors and to be without effect in cells which lack these
receptors. Exposure of cells with opiate receptors to morphine for 12 to 48 hours results in an increase in adenylate cyclase
activity which compensates for the inhibition of enzyme activity by morphine. Cells have normal cAMP levels and appear tolerant
to morphine because the increase in adenyalte cyclase activity is approximately equal to the inhibition of enzyme activity
by morphine. However, the cells 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 adenylate cyclase by narcotics accounts for the phenomena of
narcotic dependence and tolerance. The recently discovered endogenous opiate peptides, Met-enkephalin and Leu-enkephalin,
also were shown to be potent inhibitors of adenylate cyclase. The endogenous opiate peptides and narcotics act as pleiotropic
regulators of other species of receptors which are coupled to the activation of adenylate cyclase. Thus opiates alter the
perception of cells to messages.
The apparent dissociation constant of one liquid receptor complex is 6 x 10^(-11) M.
A cell line with muscarinic inhibitory acetylcholine receptors, and another with muscarinic excitatory receptors were found.
The number of receptors, the affinities of receptors for cholinergic ligands and other properties of the receptors were determined
by measuring the [3H]-quinuclidinyl benzilate binding of receptors to the muscarinic acetylcholine receptors present, results
in a transient increase in cGMP, and a profound long lived inhibition of adenylate cyclase activity.* Exposure of cells to
acetylcholine or carbachol for 24 hours markedly 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 acetylcholine receptors
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 increase in adenylate cyclase activity.
These results show that cells with a-receptors, muscarinic acetylcholine receptors, or opiate receptors can become tolerant
to and dependent upon norepinephrine, acetylcholine, and opiates, respectively, and that withdrawal of the receptor ligand
elevates intracellular cAMP levels and shifts cells to a supersensitive state with respect to other species of receptors which
activate adenylate cyclase.
Desensitization of muscarinic receptors was found to decrease the affinity of the receptor for ligands which activate the
receptor. Although the interaction of muscarinic acetylcholine receptors with agonists exhibits an apparent negative cooperativity;
the interactions between the receptor and antagonist is not a cooperative process. Both muscarinic excitatory and muscarinic
inhibitory acetylcholine receptors were solubilized and the properties of membrane found and soluble receptors were compared.
Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors. Muscarinic acetylcholine
receptors and nicotinic acetylcholine receptors were shown to be associated predominantly with the synaptic layers of the
retina. Both muscariiric and nicotinic acetylcholine receptors are synthesized before synapses-appear in the retina; however,
during the development of the retina, nicotinic acetylcholine receptors become associated predominantly with neurites in the
synaptic layers of the retina. The properties of muscarinic acetylcholine receptors were determined at different developmental
ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma cells.
Evidence for-a new type of PGE1 receptor coupled to cGMP accumulation was obtained. Cell lines with PGEl receptors coupled
to cAMP were found as well as cell lines with 2 species of PGE1 receptors, one coupled to cAMP accumulation, the other to
cGMP accumulation. PGE1 desensitizes both species of receptors but at different rates. PGF2alpha receptors coupled to cGMP
accumulation can be selectively inactivated without inactivation of PGE1 receptors and vice versa. These results show that
the coupling of PGE1 to cAMP and cGMP are clonally inherited, independently expressed, properties.
Two pathways for gamma-aminobutyric acid synthesis were found in chick embryo retina. The first pathway depends upon the
conversion of putrescine to omithine, catalyzed by omithine decarboxylase and the subsequent conversion of omithine to gamma-aminobutyric
acid. The second route of synthesis is dependent upon the conversion of glutamic acid to gamma-aminobutyric acid, catalyzed
by glutamic acid decarboxylase. The omithine decarboxylase pathway accounts for 20% of the gamma-aminobutyric acid synthesized
in retina on the 6th embryonic day but only 1% on the 18th embryonic day.
Elevation of cAMP levels in neuroblastoma cells results in an induction of ornithine decarboxylase activity. Thus neurotransmitters
which affect cAMP levels may regulate ornithine decarboxylase activity and thus may control the rate of GABA synthesis from
Previous results led to the conclusion that veratridine, batrachotoxin, and aconitine activate the action potential Na+ ionophore
by interaction with a single class of sites; scorpion venom activates the ionophore by interaction with a different class
of sites; two species of toxin bound to separate sites are allosterically coupled and interact in a cooperative manner; and
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 purified 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 app suggest that the scorpion toxin binds to a voltage sensitive component
of the Na+ ionophore that acts cooperatively in regulating ion transport activity. Binding studies with an 125I-labeled 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 inhibition 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 involved in the action potential in adult heart: a rapidly activated axon-like Na+ ionophore responsible
for the rising phase of the action potential, a slower Ca++/Na+ ionophore responsible for the plateau phase, and a K+ ionophore
responsible for the repolarization phase. Studies with specific inhibitors of the fast Na ionophore (tetrodotoxin) and the
slow Ca++/Na+ ionophore (D-600) show that the role of these two types of ionophore in beating changes during development of
the embryonic chick heart. In early embryonic hearts, the fast Na+ ionophore is present but is not required for beating.
During development in ovo or in monolayer or aggregate culture in vitro, changes in the requirement for activity of the fast
Na+ ionophore in beating are accompanied by changes in the sensitivity of the slow Na+/Ca++ ionophore to D-600. When the
slow Na+/Ca++ ionophore is able to maintain beating without participation of the fast Na+ ionophore, its sensitivity to D-600
is high whereas 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 ionphores 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 acetylcholine 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 acetylcholine receptors in embryonic
and adult heart as assessed by competition studies with muscarinic and nicotinic agents. Receptors, as detected by QNB binding,
are present in unresponsive early embryonic hearts and the number per mg heart protein does not increase dramatically during
development. Receptor desensitization is accompanied by a small change (3 fold increase) in KD for muscarinic agonists as
measured by competition with 2H QNS. This change in KD occurs only in hearts that are responsive to mucarinic agents and
thus may be associated with the physiological 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 mechanism of catabolite repression in E. coli show that glucose inhibits adenylate cyclase activity reversibly
in cells treated in toluene, and that phosphate is required for high adenylate cyclase activity 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 the phosphoenolpyruvate:sugar phosphotransferase system components were examined.
Cyclic AMP levels were normal in a 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 starvation conditions
exhibits high adenylate cyclase activity and the adenylate cyclase of this mutant is unusually sensitive to variations in
carbon source. The addition of phosphoenolpynavate 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 possibility that Enzyme I interacts with adenylate cyclase and that
the PEP-dependent phosphorylation of Enzyme I is responsible for activation of adenylate cyclase.
Levels of cGMP were compared with cAMP levels when E. coli was grown under different conditions. The results showed: 1) cells
starved for a carbon source for a short time have high levels of cAMP and low levels of cGMP. Addition of a carbon source
leads to a decrease in cAMP and an increase in cGMP, a bi-directional change. Washed cells starved for a carbon source for
long periods have low cAMP levels which do not respond to the addition of glucose; however, the addition of glucose results
in a transient increase in cGMP levels. The results reveal the presence in E. coli of reactions which inversely couple cGMP
and cAMP concentrations and show that the regulation of cGMP levels can be uncoupled from that of cAMP.
Since specific species of tRNA are involved in amino acid mediated repression and end-product inhibition, the effect of amino
acid deprivation upon tRNA of relaxed and stringent strains of E. coli was studied. In relaxed control E. coli leucine starvation
results in the formation of new isoacceptor species of leucine, histidine, arginine, valine, and alanine-specific tRNA and
quantitative changes in the concentration of some other isoacceptors. Experiments with stringent strains were the use of
uracil starvation or rifampicin addition provided evidence for the de novo synthesis of new species of isoacceptor tRNA.
The new species of leucine RNA are not aggregation artifacts, or nuclease-damaged forms of anormal leucine isoacceptor, or
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 5,6-dihydrouridine and 4-thiouridine, we think that
the biochemical explanation for the accumulation of new tRNA species under conditions 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 obtained on the mechanism of arginyl-tRNA synthetase 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 labeling experiments indicate that no enzyme bound arginyl-adenylate is formed in the absence
of added tRNA. 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 pure preparation of arginyl-tRNA synthetase from E. coli indicate that
metals may have two functional roles in the catalytic mechanism. Complete metal activation is observed when MgCl2, MnCl2,
CoCl2, or FeCl2 is present at a concentration (5 mM) in excess of the total ATP concentration (2 mM). When CaC12 is substituted
for MgC12, activity is not observed unless a small amount (0.1. mM) of MgCl2, MnCl2, CoCl2, FeC12 or ZnCl2 is added. On the
basis of these experiments, we visualize a model in which the enzyme possesses a site for free metal which, when filled, lowers
the KM for all three substrates (arginine, tRNAarg, and metal-ATP) and increases the Vmax of the reaction.