Modulation of Synapse Formation by Cyclic Adenosine Monophosphate M. Nirenberg, S. Wilson, H. Higashida, A. Rotter K. Krueger, N. Busis, R. Ray, J. G. Kenimer, M. Adler How neurons in the developing ner- vous system form synapses and distin- guish appropriate from inappropriate synapses remains one of the central, unsolved problems in neurobiology. In 1963, Sperry (I) proposed the chemoaf- finity hypothesis; namely, that neurons bear positional labels (that is, molecular addresses) that are recognized by com- plementary molecules on the synaptic target cells and thereby determine the specificity of neuronal connections. He also suggested that two gradients of mol- ecules on retina neurons at right angles to one another, which interact with com- plementary molecules on the target neu- rons in the tectum, might be a mecha- nism for matching synaptic connections and reproducing a point-to-point map of the retina in the tectum. If synapse rec- ognition molecules exist, monoclonal antibody technology should be a power- ful tool for their detection. Many investi- gators are now using this approach. Other mechanisms such as regulation of gene expression by environmental fac- tors such as hormones, neuromodula- tars, transynaptic communication, or molecules secreted by neighboring or other cells surely play important roles in the assembly of synaptic circuits. For example, Le Douarin (2) and Patterson (3) and their colleagues have shown that during development neurons from the neural crest can express either the gene for tyrosine hydroxylase, which cata- lyzes the first step in the pathway for norepinephrine synthesis, or the gene for choline acetyltransferase, which cata- lyzes the synthesis of acetylcholine, de- pending on the presence of an extracellu- lar macromolecule, purified by Weber (4), which is secreted by other cells, or the extent of depolarization of the neu- ron. In addition, Mudge (5) has shown that the expression of somatostatin. a peptide transmitter or neuromodulator. by dorsal root ganglia sensory neurons is dependent on molecules secreted b} nonneural cells. Raff et al. (6) also have shown that fetal calf serum markedly influences the differentiation pathway expressed by glial cells in the central nervous system. Edelman and his colleagues (7) discov- ered a neuronal glycoprotein rich in sial- ic acid residues, termed N-CAM (neural cell adhesion molecule), that mediates intercellular adhesion in the absence of Ca*+ and probably plays an important role in the development of the nervous system by conserving the topographic relationships between individual neuron5 or axons (or both) in a set of neurons~ even though axons may migrate lo@ distances before synapsing. Molecule5 that mediate Ca*+-dependent intercell"- lar adhesion (8) and factors that promote retina cell adhesion, such as cognin c9'. and ligand and agglutinin (IO), also ha"' been described, but little is known aho"' their function in the nervous system- Other mechanisms such as contact guiJ. ante, chemotaxis, cell survival factop' guidance of neurites by gha (II). sn' . . The authors are or have been membe;;$*~ Laboratory of Biochemical Genetics: Heart. Lung, and Blood Institute. NatIona' r: tutes of Health. Building 36. Room 1C-06. Beth Maryland 20205. ,lection for synchronous or sequential ;p,mission across two or more synap- 3j that innervate a neuron may also play ,,portant roles in synaptogenesis. ' \Ke have used monoclonal antibodies ,d cultured cell systems to study syn- ,pje formation and plasticity. Some ,,,,dies with retina cells are discussed nrst, and then studies on the plasticity of ,,.,,apses formed by clonal neuroblasto- ,&hybrid cells with striated muscle ,.clls are reviewed. A dorsal-ventral gradient of protein in ,t,ina. Trisler et al. (12) obtained a monoclonal antibody that recognizes a ~1 membrane protein distributed in a large dorsal-ventral topographic gradient ,,, chick retina (Fig. 1). The concentra- tion of antigen detected at the dorsal margin was at least 35fold higher than [hat found at the ventral margin of the retina, and the concentration of antigen detected varied continuously add loga- rithmically with the logarithm of distance &ng the circumference of the retina from ventral to dorsal poles of the gradi- ent. Thus, the protein defines a bilateral- ly symmetrical, dorsal-ventral axis of the retina and can be used as a marker of cell position in the retina with respect to the dorsal-ventral axis. The antigen, termed TOP (toponimic), was detected on all cells examined in dorsal and middle reti- na, but more TOP was detected on cells from dorsal retina than on cells from middle retina. The TOP antigen was solubilized and purified by antibody-agarose column chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel elec- trophoresis. A single band of protein was obtained with a molecular weight (M,) of approximately 47,000 (13). TOP was de- tected in optic cups of 4%hour chick embryos (14), and evidence for a gradi- ent of TOP was found in 4-day embryo retinas. A gradient therefore is generated as neurons are generated in the retina and the gradient is maintained through- out embryonic development and in the adult. Neurons first appear in the central Portion of retina and then are added in concentric, ever widening rings. Thus, central retina is the oldest portion of the retina and peripheral retina is the young- est. How a dorsal-ventral gradient is generated as the retina forms and is Perpetuated is not known. TOP was detected, in order of de- creasing concentration, in retina, cere- brum, and thalamus; little or no antigen was found in other parts of the nervous sYstem or in other tissues. Gradients of TOP were found in chicken, turkey, duck, and quail retina, but the antigen was not detected in rat, Xenopus laevis, AQfla pipiens. or goldfish retina. The antigenicity of TOP is destroyed monoclonal antibodies Fe specific for a by trypsin; however, cells dissociated single class of cells in retina such as with trypsin from dorsal, middle, or ven- photoreceptors, horizontal neurons, tral retina, cultured separately or com- Miiller cells, or ganglion neurons, or for bined in various proportions, continue to a family of cells such as those in the synthesize the antigen and accumulate inner nuclear layer of retina. Another the amount of TOP that would be expect- monoclonal antibody, A2B5 (18), recog- ed with cells from the corresponding nizes unidentified gangliosides with sial- Summary. Synapses between neuroblastoma-hybrid cells and myotubesexhibit a high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) ,levels of the hybrid cells for several days results in the appearance of functional voltage- sensitive Ca2' channels, which are requjred for evoked secretion of acetylcholine. The results show that cyclic AMP regulates synaptogenesis by regulating the 2+ expression of voltage-sensitive Ca channels, and suggest that cyclic AMP affects posttranslational modifications of some glycoproteins and cellular levels of certain proteins. region in the intact retina in ovo. Thus, the number of antigen molecules detect- ed on retina cells after 10 days in culttire depends on the prior position of the cells in the intact retina. These results suggest that the retina is composed of a gradient of cells that express different amounts of TOP, de- pending on the position of the ceils in retina along the dorsal-ventral axis of the retina. The function of TOP is not known. Monoclonal antibodies that rec- ognize an anterior-posterior gradient of molecules in retina were looked for, but were not found (15). However, the dem- onstration that TOP is a cell membrane protein and is expressed on the basis of cell position in the retina, rather than cell type, suggests that TOP may play a role in the specification of positional informa- tion in the retina. We are trying to clone complementary DNA (cDNA) corre- sponding to TOP messenger RNA (mRNA) to use to define the amino acid sequence of TOP and to explore the mechanism of regulating TOP expres- sion. Other monoclonal antibodies to reti- na. Grunwald et al. (16) showed that antibody 13H9 recognizes cell mem- brane protein detected on most or all cells in retina; however, antigen was not detected on neurons or glia in other parts of the nervous system. It is of interest to determine whether the protein specifies a compartment of cells; that is, functions as a cell adhesion molecule that enables retina cells to adhere preferentially to one another rather than to other cells. Three monoclonal antibodies recognize antigens that are restricted to the outer synaptic layer of retina (113F4, 92A2, and 18B8); another antibody (16G6) rec- ognizes antigen in both the inner and outer synaptic layers of retina. Antibody 18B8 binds to glycoproteins and uniden- tified species of gangliosides (f 7). Other ic acid residues and glycoproteins (17) that are markers of neurons and some glia (6, 18). Cultured retina ceils. Chick retina contains abundant nicotinic and musca- rinic acetylcholine receptors that mostly are distributed in layers within the inner synaptic layer of retina (19). Cultured neurons dissociated from chick embryo retina also express choline acetyltrans- ferase and acetylcholine receptors, and the neurons form approximately as many synapses in vitro (1.5 X lo9 synapses per milligram of protein) as they do in ovo, as judged by electron microscopy (20). The specificity of synapse formation by retina neurons was examined by co- culturing dissociated chick embryo or rat retina neurons with inappropriate synap- tic partner cells such as striated muscle cells that possess many nicotinic acetyl- choline receptors. Retina neurons form functional synapses with most striated muscle cells in 90 minutes, but these synapses are transient and slowly disap- pear over a period of 5 to 10 days (21- 23). Cholinergic neurons that are able to synapse with myotubes first appear in chick retina on day 6 of embryonic de- velopment, are most abundant on day 8 and comprise approximately 8 percent of the retina cell population, atid lose the ability to form synapses with.myotubes by day 16 of embryonic development (23). However, synapses between retina neurons increase during the culture peri- od and remain abundant after all synap- ses between retina neurons and muscle cells terminate. Two processes contribute to the turn- over of retina neuron synapses with myotubes. First, .retina neurons are able to form synapses with striated muscle cells only for a short time during devel- opment (23); and second, synapses be- tween retina neurons and myotubes ter- minate because retina neurons preferen- tially adhere to other retina cells rather than to myotubes (21). Preparations of neurons from chick embryo spinal cord, which presumably contain motor neurons that normally in- nervate striated muscle cells, also form synapses with cultured muscle cells, but the number of synapses remains con- stant during subsequent culture (22). Therefore, spinal cord neurons either form stable, long-lived synapses w&h muscle cells or attain a steady sf%t'e wherein the rate of synapse formation is equal to the rate of synapse termination. These results show that inappropriate synapses between retina neurons and myotubes form rapidly and are terminat- ed slowly, that synapses formed by cho- linergic neurons from retina and spinal cord turn over at different rates, and that differences in synapse turnover rates of two populations of synapses can result in the selective retention of one population and the loss of the other. Clonal Neuroblastoma Cell Lines Adult neurons do not divide; however, the establishment of clonal lines of neu- roblastoma cells from a transplantable mouse neuroblastoma tumor (C- 1300) of spontaneous origin provided a source of relatively homogeneous populations of dividing cells of neural origin (24). Char- acterization of these (24) and other (25) clonal lines of C-1300 neuroblastoma `showed that the cells have excitable membranes (26) and other neural proper- ties, and that the expression of genes for neural properties is inherited and thus can be perpetuated. Clonally inherited differences in phenotype also were found; for example, some neuroblastoma cell lines synthesize acetylcholine (25), others catecholamines; but most do not synthesize these compounds. Cells from neuroblastoma lines that synthesize acetylcholine were cocul- tured with striated muscle cells, which possess abundant nicotinic acetylcholine Table I. Cell line phenotypes [see (32, 34, 35)]. Cell ACh' lines forma- (No.) tion K+-Dependent 45Ca2' [3H]ACh uptake release Vesicles Small Large clear dense core ACh receptor aggregation protein Synapse : + +++ +++ + + + +++ + + + + + + + 2 + - + + + - 5 + ++ - + + + - or + 3 + ++ 2 + - or + 9 - - - `Acetylcholine (ACh). 2 4 6 8 5 10 50 100 Retina section Maximal distance t%) Perpendicular. Fig. 1. Geometry of the TOP gradient in 14-day chicken embryo retina (12). (A) Specifically bound `2sI-labeled F(ab'), (pmole per milligram of protein) is shown on the ordinates in (A) and (B) and within the appropriate segment of retina tested. (B) The circumference of the retina is 14.5 mm which corresponds to 100 percent on the abscissa. (A) Strips of retina from ventroanterior (0 percent) to dorsoposterior (100 percent) retina margins were removed, and each was cut into nine segments and assayed for TOP. (0) Strips of retina from anterior (0 percent) to posterior (100 percent) margins of the retina perpendicular to the choroid fissure were prepared and assayed as above; (0) data from panel A. 7% receptors, or with cardiac muscle cells that have muscarinic acetylcholine re- ceptors. However, for several years we, and others, failed to detect synapses. We thought that these cell lines might not express ah genes for proteins that might be required for synaptic communication, and therefore we fused- neuroblastoms cells with other cells and generated many somatic hybrid cell lines (26). Hybrid cell lines were found that express new neural properties not detected with parental cells (27, 28); with other hybrid cell lines some neural properties were extin- guished. Eventually five cell lines were found that synthesize acetylcholine and form many synapses with cultured myo- tubes (32, 33). The early attempts to form synapses with neuroblastoma cells failed for two reasons. (i) The extent of neural maturation and ability of cells to form synapses are regulated and are highly sensitive to environmental condi- tions, making it necessary to find condi- tions that yield populations of "differen- tiated" cells. (ii) Most, but not all. of the cholinergic neuroblastoma cell lines that were tested do, indeed, lack reactions that are required for synapse formation. Empirically, we found that populations of neuroblastoma or hybrid cells can be shifted from a poorly differentiated, syn- apse incompetent state, to a well-differ- entiated, synapse competent state, by increasing intracellular levels of cyclic adenosine monophosphate (AMP) for days. Selection for nondividing ceils also yields well-differentiated populations of cells. In Fig. 2 are shown photomicrographs of cells from four of the five cell lines that form many synapses with striated muscle cells. The NBrlO-A and NBr20-A cells originated by fusion of mouse neu- roblastoma N18TG-2 (26) with clonal BRL30-E rat liver cells, NCB-20 cells (29) resulted from fusion of N18TG-2 cells with fetal Chinese hamster brain cells, and NG108-15 (30) resulted from fusion of N18TG-2 with C6BU-I (28) rar glioma cells. Few neurites or synapse' were found when cells were in the W- rithmic phase of growth. However, es. posure of cells for 7 days to I mM dib". tyryl cyclic AMP, which promotes nrur. ite extension, and to 1 percent (rather than 5 percent) fetal bovine s;lzs' which reduces neurite retraction. . ' cells with neurites that can be more thnn 2 mm in length (31). Other cell lines have high concen"' tions of acetylcholine, adhere Wei' " myotubes, but do not form sYz:z,; (32). A summary of phenotypes lines with or without synaptic defects " shown in Table 1. The NBrlO-A. NBBr3" A, FJCB-20, NG108-15, and NS-26 neu- &astoma cells (25) form many synap- ,gs with cultured myotubes (32,33), syn- ,hzsize acetylcholine (32), have function- .,I "oItage-sensitive CaZf channels (34), have small clear vesicles approximately 4 nm in diameter and large dense-core ,,,sicIes 180 nm in diameter (41), and +se acetylcholine into the medium Joj a protein that stimulates the aggrega- I,on of nicotinic acetylcholine receptors on myotube plasma membranes (35). Cells from three lines take up Ca'+ ions jlo~Iy (34) and secrete little acetylcho- line when depolarized by 80 mM K' ions, and form few synapses with muscle ce~~~, Cells from two lines lack functional ,oltage-sensitive Ca2+ channels (34) and ~0 not form synapses. Cells from five lines take up Ca2+ when depolarized by K- ions but do not respond by secreting more acetylcholine (32), and few or no synapses were found. These cells lack a ca?+-dependent acetylcholine secretion reaction (or reactions); however, acetyl- choline is secreted into the medium in the basal, unstimulated state. Cells from [hree lines have small clear vesicles but lack large dense-core vesicles and func- tional protein that induces nicotinic ace- tylcholine receptor aggregation on myo- tube membranes (35), and form few or no synapses. Nine additional cell lines have little or no choline acetyltransferase ac- tivity, and therefore they synthesize lit- tle or no acetylcholine (32) and do not form functional synapses with striated muscle cells. Regulation of synaptogenesis. Thus far. we have identified 12 species of receptors that are expressed by NGlOS- IS cells, including receptors for prosta- glandin E, (PGE,) (36,37), prostaglandin Fz (PGF2) (36). adenosine (38), Met- enkephalin (36), alpha-Zadrenergic re- ceptors (39), depolarizing muscarinic acetylcholine receptors (40), serotonin and LSD receptors (29), and receptors for bradykinin, neurotensin, angiotensin II. and somatastatin (32), and have de- fined cell responses to the ligands for these receptors. Some receptors, such as those for PGE,, mediate activation of adenylate cyclase; other receptors such as Met-enkephalin receptors, muscarinic depolarizing acetylcholine receptors, and alpha-2-adrenergic receptors medi- ate inhibition of adenylate cyclase. Increase of cyclic AMP in neuroblas- toma or hybrid .cells for 5 to 7 days, obtained either by treating cells with PGE, to increase the endogenous rate of Cyclic AMP synthesis or by inhibition of Cyclic nucleotide phosphodiesterase with dibutyryl cyclic AMP, or theophyl- line. resulted in increases in the percent- Table 2. Effect of culture conditions on synap- togenesis and acetylcholine secretion by NGlOS-15 cells. Each value is the mean of values obtained from more than 75 myotubes. [Data from (32)] Culture conditions Myo- Syn- tubes aptic with re- syn- sponse apses fre- (%) quency* Control 1 mM dibutyryl cyclic AMP 1 mM theophylline 10 m PGE, lOpMPGE, + 1mM theophylline IS 0.7 55 14 64 10 63 II 98 32 *The number per minute per myotube. age of myotubes tested that were inner- vated and the rate of spontaneous secre- tion of acetylcholine from NG108-15 cells at synapses (32) (Table 2). Presum- ably, each depolarizing response of a myotube to acetylcholine is due to the spontaneous secretion of acetyicholine from a single NG108-15 vesicle. NG108- 15 cells and myotubes were cocultured and treated for 5 to 7 days with the compounds shown; then myotubes were assayed for synapses by intracellular mi- croelectrode recording. Treatment of cells with 1 mM dibutyryl cyclic AMP, 1 mM theophylline, or 10 PM PGE, in- creased the percentage of muscle ceils tested that were innervated from 15 to approximately 60 percent and increased 14- to 20-fold the frequency of spontane- ous synaptic responses of myotubes (the miniature end-plate potential frequency). Treatment of cells with 10 PM PGE, and 1 mM theophylline resulted in innerva- tion of 98 percent of the myotubes tested and increased the frequency of synaptic responses of myotubes 45-fold. No im- mediate effect of these compounds on the cell membrane potential or rate of acetylcholine secretion was detected. Half-maximal ingreases in synapses and rate of sfiontaneous acetylcholine secre- tion at synapses were observed when cellular cyclic AMP levels were in- creased for 1 to 2 days; maximal in- creases were obtained when cells were treated for 3 to 5 days (32). In other experiments, NGIOS-15 cells were incubated with PGE,, theophylline, dibutyryl cyclic AMP, or PGE, and theo- phylline for 5 to 7 days: then the com- pounds were withdrawn and cells were incubated for an additional 4 to 14 days to determine whether the effects on syn- apses and acetylcholine secretion were reversible. On withdrawal of the com- pounds, synapses and acetylcholine se- cretion gradually returned to control val- ues in 7 to 11 days (32). Thus, the effects of the compounds on synapses are ex- pressed slowly and are long-lived. Cyclic AMP levels of NG108-15 cells increase markedly in the presence of 10 PM PGE, and 1 mM theophylline and Fig. 2. Neuroblastoma hybnd cells from lines that form many synapses with cultured myotubes were treated for 7 days with 1 r&f dibutyryl cyclic AMP and the concentration of fetal bovine serum was reduced from 5 to 1 percent between day 5 and day 7. The bar in the lower right hand panel corresponds to 50 km in each panel. [Data from (32)] 797 remain higher than those of control cells for seven or more days. `Intracellular acetylcholine in NG108-15 cells also in- creases eight- and threefold when cells are treated for 3 days with PGE, and theophylline or dibutyryl cyclic AMP, respectively (32). NGlOS-15 cells treated with dibutyryl cyclic AMP (41) or PGE, and theophylline (32) for five or more days contain many large dense-core vesi- cles and small clear vesicles, whereas control cells contain few vesicles. The cyclic AMP-dependent increase in intra- cellular acetylcholine is due, at least in part, to an increase in the number of acetylcholine storage vesicles in cells. Depolarization of NG108-15 or NBrlO- A cells with 80 mM K+ ions, in place of 80 mM Na+ ions, has no effect on the rate of acetylcholine secretion bjl un- treated NG108-15 or NBrlO-A cells. However, cells gradually are shifted from an unresponsive to a responsive state with respect to depolarization-de- pendent secretion of acetylcholine when treated for 5 to 7 days with 1 mM dibu- tyryl cyclic AMP or 10 pM PGE, and 1 mM theophylline. Half-maximal and maximal increases in acetylcholine se- cretion due to cell depolarization were obtained when NG108-15 cells were treated with 1 mM dibutyryl cyclic AMP for 2 and 5 days, respectively (42). Depolarization of nerve terminals is known to activate voltage-sensitive Ca2+ channels; Ca2+ ions then flow into the cytoplasm of axon terminals and in- crease the rate of secretion of transmitter at the synapse. We therefore examined the effect of prolonged elevation of cy- clic AMP levels of NBrlO-A or NGlOS- 15 cells on voltage-sensitive Ca*+ chan- nel activity. Four kinds of assays were used (34). 45Ca2+ flux, net uptake of Ca*' by cells was measured with a Ca*+ specific electrode, Ca2' fluxes were de- termined in the presence of murexide by a spectrophotometric assay with a stopped-flow apparatus, and Ca" action potentials of cells were assayed by intra- cellular microelectrode recording. We found by each method of assay that logarithmically dividing cpntrol cells have little or no voltage-sensitive Ca*+ channel activity; however, prolonged el- evation of cellular cyclic AMP activation of adenylate cyclase of cells with PGE, , or by inhibition of cyclic nucleotide phosphodiesterase with dibutyryl cyclic AMP or theophylline, gradually results in the acquisition of functional voltage- sensitive Ca2+ channels by cells. Assay of Ca*' action potentials elicited by elec- trical stimulation of single cells with in- tracellular microelectric recording showed that most untreated NG108-15 or NBrlO-A cells lack functional voltage- sensitive Ca*+ channels. However, Ca" with PGE, and theophylline or dibutyryl action potentials were found in 100 per- cyclic AMP for 2 and 4 days, respective- cent of the cells tested that had been ly. treated for four more days with dibutyryl Relatively weak voltage-sensitive cyclic AMP. Ca2+ channel activity appears in untreat. As shown in Fig. 3, 45Ca2+ uptake by ed NBrlO-A cells when cells form co,,- logarithmically dividing, control NBrlO- fluent monolayers. Thus, cell concentra. A ceils is not affected by depolarization tion or adhesive interactions between of cells with 80 mM K+. However, cells cells also regulates the expression of that had been treated for 7 days with 10 voltage-sensitive Ca'+ to some extent. pM PGE, and 1 mM theophylline or Nitrendipine and other dihydropyc. with 1 mM dibutyryl cyclic AMP re- dine derivatives inhibit voltage-sensitive spond to depolarization by 80 mM K+ with a rapid influx of 4'Ca2+ via voltage- Ca2+ channels of smooth muscle (43), striated muscle (44), and cardiac muscle sensitive Ca*+ channels (34). Depolar- (45), and specific binding sites for 3H- ization-dependent 45Ca2+ uptake is in- labeled nitrendipine have been found in hibited completely by 1 X 10e4M D-600 these tissues and in brain (46). The ni- (half-maximal inhibition was obtained trendipine receptors are thought to be with 9 X lo-`M D-600), an alkaloid part of the voltage-sensitive Ca2+ chan- known to inhibit voltage-sensitive Ca2+ nel complex, perhaps functioning as reg channels and slow Na+ channels. 45Ca2+ ulators of channel activity. uptake also is inhibited by La3+, Co'+, Kongsamut and Miiller have shown and Ni*+ ions. that 45Ca2+ uptake by NG108-I5 cells Exposure of NG108-15 cells to PGE, mediated by voltage-sensitive Ca2+ increases cellular cyclic AMP levels channels is inhibited by nitrendipine within seconds: however, no immediate (47). We have confirmed this and find effects of PGE,, PGE, and theophylline, that NBrlO-A cells are inhibited half- or dibutyryl cyclic AMP on voltage-sen- maximally by 3 nM nitrendipine. A sin- sitive Ca2+ channel activity were detect- gle class of specific binding sites for 3H- ed. Half-maximal and maximal voltage- labeled nitrendipine was found in mem- sensitive Ca2' channel activity were ex- branes from NBrlO-A cells that had been pressed by cells that had been treated treated with PGE, and theophylline with a dissociation constant, estimated by Scatchard analysis, of 2 x lO-"M, which is similar to values reported for POE, o theophylline o other tissues (43, 4546). The maximum number of specific nitrendipine binding sites was estimated to be 61 fmole per milligram of NBrlO-A membrane pro- tein, which is equivalent to approximate- ly 16,000 specific sites for nitrendipine per cell. In contrast, few or no specific binding sites for 3H-labeled nitrendipine were detected in membranes from un- treated, logarithmically dividing NBrlO- A cells. These results show that cyclic AMP regulates the number of specific nitrendipine receptors per cell. Specific binding sites for 3H-labeled nitrendipine Control cells also were not detected in membranes prepared from two lines of hybrid cells 0 2 4 8 0 10 Time (minuted (SB21B-1 and SB37-B) that lack func- tional voltage-sensitive Ca2+ channels Fig. 3. The effect of culture conditions on the expression of functional voltage-sensitive and do not synapse with muscle cells. Ca'+ channels of NBrlO-A cells. Uptake of Cyclic AMP increases the probabilitY "Cat' due to activation of voltage-sensitive of opening Ca*+ channels of cardiac Ca" channels of untreated logarithmically muscle cells (48); however, responses t0 dividing control NBrlO-A cells, cells cultured cyclic AMP are rapid and thus differ for 6 days with 1 mit4 dibutyryl cyclic AMP, or 10 @I4 PGE, and 1 m&I theophylline. The from the slow effects found with NBrlO- cells were depolarized with 80 mM K' (in A cells. place of 80 ITIM Na+). Values for +Za'+ The molecular weights of nitrendipine binding to cells or uptake at 5.4 m&I K', receptors in intact membranes of smooth which were not inhibited by 100 m D-600 and were not mediated by voltage-sensitive muscle (49), transverse tubule mefl Ca" channels, were subtracted from the val- branes of skeletal muscle, and cerebral ues shown. Uptake of Ca*' dependent on cell cortex synaptic membranes (44) Were depolarization was completely inhibited by estimated by radiation inactivation tar@' 100 p& D-600. [Data from (34)] analysis to be 278,000, 210,000, and 210,000, respectively. Available infor- mation suggests that the nitrendipine re- ceptor complex is a glycoprotein with N- acetylglucosamine or sialic acid residues (or both) (50). Nitrendipine receptors of smooth and cardiac muscle were report- ed to be covalently labeled with a radio- active affinity label analog of nitrendi- pine, 3H-labeled 2,6-dimethyl-3,5-di- carbomethoxy - 4 - (2 - isothiocyanato- phenyl)- 1,4-diiydropyridine; labeled pro- tein then was solubilized and fractionat- ed. A peak of labeled protein with a molecular weight of 45,000 was identi- fied (49). These results suggest that the molecular weight of voltage-sensitive Ca*+ channel in membranes is 210,000 to 278,000, that each channel is composed of two or more subunits, and that one subunit is a protein with a molecular weight of 45,000, which binds nitrendi- pine. NG108-15 cells that had been grown with or without 10 m PGE, were incu- bated with ["Slmethionine to label the protein; the 35S-labeled glycoproteins then were solubilized and fractionated by wheat germ agglutinin-, ricin-, or len- til-lectin column chromatography and by two-dimensional gel electrophoresis (51). Elevation of cellular cyclic AMP levels resulted in the disappearance of some 3'S-labeled glycoproteins, the ap- pearance of new 35S-labeled glycopro- teins with different molecular weights, changes in the apparent abundance of some 35S-labeled glycoproteins, as well as changes in the isoelectric points of other 35S-labeled glycoproteins. A "S- labeled glycoprotein with a molecular weight of approximately 45,000 was elut- ed from wheat germ agglutinin-sepha- rose with N-acetylglucosamine was ob- tained from cells with high cyclic AMP levels, but was not detected in untreated cells. Twelve "S-glycoproteins were de- tected that were expressed by NG108-15 cells with high cyclic AMP levels but not by control cells, and many other )$- labeled glycoproteins were obtained from PGE,-treated cells with radioactiv- ities 2.5- to IO-fold higher than those of control cells. These results extend previ- ous reports of differentiation-specific changes in neuroblastoma proteins (52). Exposure of neuroblastoma or hybrid cells to dibutyryl cyclic AMP alters the levels of some species of polysomal mRNA (53). Polysomal polyadenylated (~01~ A+) RNA from "undifferentiated" and "differentiated" neuroblastoma cells were compared; many species of Polysomal poly A' RNA were found in RNA from undifferentiated cells, but not differentiated cells (54, 55), and con- versely, many species of poly A+ RNA were expressed by differentiated neuro- blastoma cells that were not expressed by undifferentiated cells (55). In prokaryotic cells (cyclic AMP * ca- tabolite activator protein) complexes bind to certain sites on DNA and thereby regulate the initiation of transcription of certain genes. Cyclic AMP also regulates the levels of some species of mRNA and protein in eukaryotic cells (56), but rela- tively little is known about the mecha- nisms of regulation. Cyclic AMP mark- edly increases the expression of many neural properties in neuroblastoma or hybrid cells, such as voltage-sensitive channels for Ca2', Naf, and KC, and also Ca*+-dependent K' channels, neur- ite extension, vesicles, synapses, acetyl- cholinesterase, and with some cell lines, choline acetyltransferase, or tyrosine hy- droxylase activities. We find that cyclic AMP regulateg synaptogenesis, at least in part, by regulating the expression of voltage-sensitive Ca*+ channels, which are required for stimulus-dependent se- cretion of transmitter at synapses. The results suggest that cyclic AMP affects posttranslational modifications of some species of glycoprotein. Appropriate cloned cDNA probes are needed to de- termine whether cyclic AMP affects the levels of some species of mRNA and to define further the cyclic AMP-depen- dent mechanisms that affect synapse for- mation and plasticity. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. Z: 12. 13. 14. 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