"Annual Report of the Laboratory of Biochemical Genetics" [summary of laboratory projects]
This annual report includes a summary of research undertaken in the Laboratory of Biochemical Genetics and specific descriptions
of two projects listing Nirenberg as the principal investigator: 1) Receptor Mediated Regulation of Adenylate Cyclase; and
2) Cell Recognition and Synapse Formation. Major findings, significance to biomedical research, and associated publications
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1979-09 (September 1979)
Nirenberg, Marshall W.
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1978 - September 30, 1979 (September 1979)
Annual Report of the Laboratory of Biochemical Genetics
National Heart, Lung, and Blood Institute
October 1, 1978 through September 30, 1979
Biochemistry of Synaptogenesis. Clonal lines of hybrid cells derived by fusion of neuroblastoma cells with other cell types
were shown previously to form synapses with striated muscle cells with high frequency. The formation of synapses between clonal
cells of neural origin, such as NBr10A or NG108-15 hybrid cells, and rat striated muscle cells was found to be regulated.
Exposure of hybrid cells for 3-7 days to PGE1, which results in activation of adenylate cyclase, or exposure to various cyclic
nucleotide phosphodiesterase inhibitors markedly increases the number of synapses formed. The effects of putative neurotransmitters
or hormones on intracellular cyclic AMP or cyclic GMP levels, voltage-sensitive Ca2+ channel activity, and acetylcholine secretion
were determined. Receptor-mediated increases in intracellular cyclic AMP or cyclic GMP levels had no immediate effect on K+-dependent
45Ca2+ uptake by cells or on acetylcholine secretion from cells. However, prolonged exposure of hybrid cells to PGE1 results
both in an increase in cellular cyclic AMP and the gradual acquisition by cells of functional voltage-sensitive Ca2+ channels.
Concomitantly cells acquire the ability to secrete acetylcholine in response to a depolarizing stimulus and can then form
functional synapses with muscle cells. These results show that the acquisition of voltage-sensitive Ca2+ channels is regulated
and that this reaction in turn controls the formation of synapses.
D600 inhibits 45Ca2+ uptake dependent on 80 mM K+ (IC50 = 2 x 10-7 M), but has little or no effect on 45Ca2+ uptake in the
presence of 5mM K+. 45Ca2+ uptake also is inhibited by 10 mM La3+, Co2+, Ni2+, Mn2+, Sr2+, or Ba2+, but not by 10 uM tetrodotoxon,
20 mM tetraethylammonium, or 1 mM 3,4-diaminopyridine.
Other cell lines were found that synthesize acetylcholine but do not form synapses with striated muscle cells. Various types
of synapse defects were detected; including defects in voltage-sensitive 45Ca2+ channels, vesicles, and an additional unidentified
reaction that is required for acetylcholine secretion.
To identify molecules required for synaptogenesis or communication across the synapse, hybrid cell lines which synthesize
mono-specific antibodies were obtained by fusion of clonal myeloma cells with spleen cells immunized against cells from the
nervous system. Some of the hybridoma cell lines that were obtained synthesize monospecific antibodies of high titre directed
against membrane antigens found on some cells from the nervous system that were not detected with cells from other tissues.
One of these cell lines, A2B5, synthesizes antibody directed against an antigen that was shown by indirect immunofluorescence
to be associated with plasma membranes of most, or all, neuron cell bodies in chick retina; however, the antigen was not detected
on axons or dendrites of neurons, on retina Muller cells, or pigment cells, or on cells from non-neural tissues.
Antigen A2B5 activity is relatively stable at 100 degrees C, is insensitive to trypsin, exhibits the solubility properties
of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a tetrasialo
GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition, 0.2 uM), or N-acetylneuraminic acid (half-maximal
inhibition, 5,000 uM), but not by other purified gangliosides tested. These results suggest that the antigen is a GQ ganglioside
in plasma membranes of retina neuron cell bodies but not membranes of axons or dendrites.
A solid-phase 125 I-Protein A radioassay for anti-cell surface antibodies was devised which employs target cell monolayers
cultured on fenestrated polyvinyl chloride 96-well plates ("transfer plates"). The calibrated aperture in the bottom
of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to
enter the wells when transfer plates are lowered in receptacles containing washing buffer or test sera. To assay for antibodies
directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with
corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of
hybrid cell colonies for production of antibodies with desired specificity.
Methyltransferases can be inhibited by S-adenosyl homocysteine or by analogs which either increase S-adenosyl homocysteine
levels or inhibit methyltransferases directly such as 3-deazaadenosine(DZA), adenosine-2',3'-diazido-5'-carboxamide
(744-99), 5'-deoxy-5'-isobutylthioadenosine(SIBA), and 5'-deoxy-5'-isobutylthio-3-deazaadenosine (DZ-SIBA).
In collaboration with P. Chiang and G. Cantoni the effects of these and other compounds on synapses between dissociated chick
embryo retina neurons and cultured rat striated muscle cells were investigated. The frequency of spontaneous synaptic responses
of muscle cells was markedly reduced by these compounds; half-maximal inhibition was obtained with 1.5 x 10-6 M DZ-SIBA, 1.5
x 10-5 M DZA, 3 x 10-5 M SIBA, or 1 x 10-4 M 744-99. Homocysteine thiolactone, 5-deoxy-adenosine, or tubercidin, which do
not in- crease levels of S-adenosine homocysteine or inhibit methyltransferase activity, do not affect the frequency of spontaneous
synaptic responses of muscle cells. These results suggest that a transmethylation reaction may be required for acetylcholine
secretion or vesicle cycling in synaptic terminals of neurons.
Regulation of Adenylate Cyclase of Cell Lines From The Nervous System. The inhibition of adenylate cyclase by morphine and
the gradual increase in adenylate cyclase activity that results when NG108-15 cells are incubated for 12 or more hours in
the presence of morphine was previously proposed as a model for the analgesic action of opiates and for the phenomena of opiate
dependence and tolerance. We now find that linoleic acid or serum lipids are required for the morphine-dependent increase
in adenylate cyclase activity, but not for inhibition of the enzyme. Similar results were obtained with norepinephrine which
activates a-receptors of NG108-15 cells, In this model system, therefore, the inhibition of NG108-15 adenylate cyclase by
morphine or norepinephrine can be dissociated from the acquisition of dependence upon opiates or norepinephrine.
Ten uM morphine or norepinephrine completely inhibit the activation of adenylate cyclase by Ca2+ ions, but inhibit basal or
PGE1-activated adenylate cyclase by no more than 55 percent in NG108-15 homogenates. The extent of inhibition of adenylate
cyclase by morphine or norepinephrine thus is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase
molecules that are activated by Ca2+ ions.
Activation of serotonin receptors of NG108-15 or NCB-20 hybrid cells by serotonin results in cell depolarization, action potentials,
and secretion of acetylcholine into the medium. These responses desensitize in less than 15 sec and are not inhibited or mimicked
by LSD. Serotonin also stimulates adenylate cyclase activity of NCB-20 hybrid cells, but the effect of serotonin does not
desensitize. Eadie-Scatchard analysis suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity.
The Hill interaction coefficient is 1.0, indicating independent, noncooperative reactions. LSD activates adenylate cyclase
(Kact = 12 nM) and also inhibits the activation of the enzyme by serotonin (Ki = 10 nM). In addition, mianserin and cyproheptadine
inhibit serotonin activation of adenylate cyclase K1 = 43 nM and 95 nM, respectvely) and LSD activation of adenylate cyclase
(Ki = 100 nM and 64 nM, respectively). These results show that serotonin and LSD interact during activation of adenylate cyclase.
Binding sites for [3H]LSD were detected in NCB-20 homogenates; the Kdapp was 36 nM, the Hill coefficient was 1.0, and the
receptor concentration was 385 fmol/mg of protein. [3H]LSD was displaced by serotonin (Ki = 110-180 nM). These results agree
well with those found to be mediated by a serotonin receptor responsive to LSD that mediates activation of adenylate cyclase.
Two binding sites for [3H]serotonin were detected in NCB-20 homogenates [Kdapp = 200 nM and 3750 nM] and serotonin-LSD interactions
also were detected.
We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, one coupled to activation of adenylate cyclase,
the other to cell depolarization and acetylcholine release; that activation of adenylate cyclase does not affect the rate
of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intracellular levels
of cAMP or cGMP in the hybrid cells tested.
Muscarinic Acetylcholine Receptors. [3H]-Quinuclidinyl-benzilate (QNB) was used to study muscarinic acetylcholine receptors
in NG108-15 membrane preparations. The apparent dissociation constant of [3H]-QNB is 1 x 10-10 M; the average NG108-15 cell
possesses 30,000 specific sites for [3H]-QNB. Activation of the receptors with acetylcholine or carbachol results in cell
depolarization, a small increase in cellular cGMP, and inhibition of adenylate cyclase. Cell depolarization and rise in cGMB
levels desensitize in 30 sec; whereas, the inhibition of adenylate cyclase does not desensitize. Scatchard analysis revealed
only one homogeneous class of [3H]-QNB binding sites; however, biphasic rates of [3H]-QNB association with and dissociation
from receptors were found. Evidence was obtained for the formation of a dissociable [[3H]-QNB-Receptor] complex which then
is converted to a form which dissociates only slowly. Hill coefficients of approximately 1.0 were found for receptor antagonists
and approximately 0.5 for receptor activators. A sequential series of reactions were proposed to account for these observations
and for the various states of the muscarinic acetylcholine receptor that were detected.
Nicotinic Acetylcholine Receptors. An aBT-horseradish peroxidase conjugate was used to study the distribution of nicotinic
acetylcholine receptors in developing chick retina. Incubation of the retina in vitro with the conjugate allowed quantitative
comparison of developmental stages. aBT-binding synapses were found at the early stages of synapse formation and comprised
between 5 and 11% of the inner plexiform layer synapse population during in ovo development.
The acetylcholine receptor aggregation factor from neuroblastoma x glioma hybrid
cells was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectric focusing. Factors
with similar activity were detected in embryonic brain and cultures of sympathetic ganglion neurons and spinal cord neurons,
but not in liver, adult brain, or embryonic glial cell cultures.
Detergent treatment under appropriate conditions removed most lipid and soluble
protein from cultures skeletal muscle cells, but left the cytoskeleton and bound components intact. This extraction was used
to distinguish tightly bound and loosely bound populations of acetylcholine receptors, which may be correlated with the degree
of receptor aggregation.
Endorphin Synthesis and Secretion. AtT-20 mouse pituitary tumor cells were shown to synthesize and secrete B-endorphin (B-lipotropin61-91).
The cells contain at least 1 nmole B-endorphin equivalents of opioid peptides per mg cell protein. Analysis of cell extracts
by gel filtration and high pressure liquid chromatography indicate that the activity is due to B-endorphin, a-endorphin (B-lipotropin61-76),
and y-endorphin (B-lipotropin61-77), in the approximate proportions 70%, 24%, and 6%, respectively. Subcellular fractionation
indicated that most of the activity is located in the granular fraction. Electron microscopy revealed the presence of osmiophilic
granules resembling the secretory granules of corticotrophs of the anterior pituitary. These granules were positive for B-endorphin/B-lipotropin
immunoreactivity, when assayed. Thus, AtT-20 tumor cells possess a mechanism similar to that of normal endocrine cells for
packaging peptides destined for secretion.
In the absence of serum, basal secretion of B-lipotropin/B-endorphin immunoreactivity is 20-30 pmoles per mg protein per hr
and secretion is linear for at least 12 hr. Fifty to 70% of the immunoreactivity secreted is due to B-linotropin-like peptides
and the rest to B-endorphin-like peptides. Thus much B-lipotropin is secreted with further processing.
Secretion is stimulated 5-8 fold by brief exposure of cells to elevated K+ ion
concentration; this stimulation is dependent on Ca++ ions. Glucocorticoids, such as dexamethasone, reduce the secretion of
B-lipotropin/B-endorphin within 2 hr; for example, secretion is reduced by 33% or 67% after 2 or 8 hrs, respectively. During
this time the intracellular content remains the same, however, intracellular content diminishes after 24 hr of treatment.
The half-maximally effective dexamethasone concentration is 2 nM. The effect of dexamethasone on secretion is abolished by
cycloheximide or actinomycin D which inhibit protein synthesis and RNA synthesis, respectively. This suggests that glucocorticoids
act at the transcriptional level to induce the synthesis of protein(s) which inhibit the secretion of ACTH and B-endorphin.
Other workers have shown recently that corticotropin/S-lipotropin mRNA is gradually reduced by glucocorticoid treatment for
1-4 days. The present results with AtT-20 cells suggest that glucocorticoids have an earlier different effect on secretion
than the slower reduction in mRNA for the prohormone.
Cyclic Nucleotides In E. Coli. Our previous studies led to the development of a model for the regulation of adenylate cyclase
involving the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The proposal has been made that Enzyme I of the
PTS interacts in a regulatory sense with the catalytic unit of adenylate cyclase:
The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state
of 'adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity
state of adenylate cyclase. Evidence in support of the proposed model
involves the observation that Enzyme I mutants have low cAMP levels and that
PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase. Kinetic studies indicate that
various ligands have opposing
effects on adenylate cyclase. While PEP activates the enzyme, either glucose
or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity provide further support
for the model outlined above.
The interaction of adenylate cyclase with sugars that are transported by systems other than the PTS also were explored. Sugars
such as lactose are transported without modification by a mechanism involving proton cotransport; this mechanism requires
a proton motive force across the cell membrane. We have been able to show that uptake of sugars through the lactose transport
system results in inhibition of adenylate cyclase activity if the proton symport mechanism is also active. The protonophore
carbonyl cyanide m-chlorophenylhydrazone also inhibits adenylate cyclase activity. These data suggest that the steady-state
electrochemical proton gradient regulates the activity of adenylate cyclase. We propose that sugar-dependent inhibition of
adenylate cyclase activity may occur by either of two mechanisms. Sugars transported by the PTS inhibit adenylate cyclase
activity by dephosphorylation of a regulatory protein, while sugars transported by the proton motive force system inhibit
adenylate cyclase activity as a result of collapse of the proton electrochemical gradient.
Metabolism of Thyrotropin Releasing Hormone. Previously we described an enzyme (pyroglutamate aminopeptidase) in brain extracts
that converts TRH to histidyl-prolineamide which spontaneously cyclizes to histidyl-proline ketopiperazine.
We also presented evidence of the presence in hypothalamic extracts of an enzyme (TRH deamidase) that converts TRH to pryoglutamyl-histidyl
proline. Further studies have led to the isolation from brain of an imidopeptidase for histidyl-prolineamide not previously
described. The enzyme was found in extracts of porcine brain acetone powder and purified by conventional column chromatography
on DEAE cellulose resulting in the separation of the enzyme from other enzymes that metabolize TRH. The best substrates for
the imidopeptidase contain an a-amino group on histidine and a blocked carboxyl group on proline, as is found in histidyl-prolineamide.
Other polypeptide hormones were shown to inhibit imidopeptidase activity. Inhibition of the enzyme by adrenocorticotropic
hormone. (l-24) is noncompetitive. These studies have led us to propose that pituitary hormones may stimulate the production
of histidyl-proline diketopiperazine by inhibiting alternate routes of TRH metabolism.
The Biological Activity of Histidyl-Proline Diketopiperazine. Previously we showed that injection of radioactive TRH into
rat brain led to the formation of radioactive histidyl-proline diketopiperazine, establishing this compound as a naturally
occurring brain peptide. While TRH could antagonize the effects of ethanol in inducing sleep in rats, the dipeptide diketopiperazine
was substantially more active than TRH. We therefore suggested that the activity of TRH in antagonizing ethanol narcosis
may require its conversion to histidyl-proline diketopiperazine.
We have continued to explore the biological activities of histidyl-proline diketopiperazine and find that it plays a role
in thermoregulation and in the
regulation of brain cyclic nucleotide levels.
Intraventricular administration of histidyl-proline diketopiperazine to rats produces a dose-dependent hypothermia at 4 degrees
or 24 degrees, but not at 31 degrees. At 4 degrees administration of TRH elicits a dose-dependent hypothermia up to 0.1 umole/Kg
which is not evoked at higher doses. At 24 degrees, TRH administration results in no change in temperature, whereas it induces
hypothermia at 31 degrees. At 4 degrees, TRH antagonizes and TRH antiserum potentiates the hypothermic effects of histidyl-proline
diketopiperazine, suggesting opposing effects of TEUJ and histidyl-proline diketopiperazine on thermoregulation.
Intraperitoneal administration of thyrotropin releasing hormone (50 umole/Kg)
produced an approximately 2-fold increase in rat brain cGMP concentration with-
in 15 min. Histidyl-proline diketopiperazine produced a similar effect, but the response was faster and shorter-lasting.
Intraperitoneal administration of ethanol (1.5 g/Kg) decreased brain cGMP concentration approximately 50% within 10-15 min;
thyrotropin releasing hormone or histidyl-proline diketopiperazine, injected 5 min after ethanol, antagonized the ethanol-induced
increase in cGMP.