Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N⁶, O^2′-dibutyryl cyclic AMP (Bt₂cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N⁶,O^2′-dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N⁶,O^2′-Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N⁶,O^2′-dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Effects of dexamethasone and dibutyryl cyclic AMP on polyamine synthesizing enzymes in mouse lymphoma cells

S49.1 Lymphoma cells were arrested in G₁ phase of the cell cycle when treated with either 1 μM dexamethasone (Dex) or 0.5 mM N⁶, O²-dibutyryl cyclic adenosine 3′ :5′ -monophosphate (Bt₂cAMP) plus 0.2 mM theophylline. However, the two agents had markedly different effects on aspects of polyamine and cyclic nucleotide metabolism within the arrested cells. Bt₂cAMP had an early and pronounced inhibitory effect on ornithine decarboxylase (ODC) activity causing a decrease to 40% of control within 1 h. However, there was no significant inhibition of ODC activity in the Dex-treated cells until 4 h of exposure, at which time ODC activity was reduced to approximately 60% of the control value. Sadenosyl-L-methionine decarboxylase (SAMD) activity was reduced by both agents, Bt₂cAMP having the more pronounced inhibitory effect. The activity of SAMD was reduced to 40% of control after 10 h of Dex, whereas Bt₂cAMP reduced the activity to approximately 25% of control within 4 h. Intracellular polyamine pools were decreased rapidly in Dex-treated cells but not in those exposed to Bt₂cAMP. Bt₂cAMP decreased the amount of type I (PK_I) and type II (PK_II) cyclic AMP-dependent protein kinase (cAMP-PK) activity to 30% of control or less within 2 h. In contrast, Dex had very little effect on either PK_I or PK_II until 24 h, when cell viability was affected. The specific activity of both PK_I and PK_II remained significantly decreased in cells exposed to Bt₂cAMP for 6 h and then resuspended in fresh medium. The rapid decrease in ODC activity in response to Bt₂cAMP and the slow recovery after washout may be due to the marked decreases in total PK_I and PK_II activities. Dex, which had no effect on PK_I and PK_II specific activities, only slowly inhibited ODC activity and recovery of enzyme activity was rapid upon resuspension in fresh medium. These data further stress the importance of the maintenance of the cellular protein kinase pools in the regulation of the recovery time to growth inhibition in response to naturally occurring steroids and second messengers.     

Human dermal microvascular endothelial cells in vitro: Effect of cyclic AMP on cellular morphology and proliferation rate

Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitor. In the presence of 5 x 10^?4 M dibutyryl cyclic AMP (Bt₂cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of control values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt₂cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt₂cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methyl-xanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt₂cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt₂cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.     

Characteristics of45Ca uptake stimulated by high KCl of differentiated and undifferentiated NG108-15 and PC12h cells

The characteristics of KCl-stimulated⁴⁵Ca uptake by neuroblastoma x glioma hybrid NG108-15 cells induced to differentiate with dibutyryl cAMP (Bt₂cAMP) and of PC12h pheochromocytoma cells induced to differentiate with nerve growth factor (NGF) were studied. The extent and rate of KCl-stimulated⁴⁵Ca uptake by differentiated NG108-15 cells induced with Bt₂cAMP were significantly higher than those of the undifferentiated cells. However, differentiation of PC12h cells induced with NGF did not enhance their extent or rate of KCl-stimulated⁴⁵Ca uptake. The effects of Ca agonist and antagonists indicated that the characteristics of KCl-stimulated⁴⁵Ca uptake by Bt₂cAMP-treated NG108-15 cells and NGF-treated PC12h cells mainly reflected those of peripheral L-type voltage-sensitive calcium channels activated by high KCl. These results suggest that differentiated neural cells did not all show an enhanced capacity for KCl-stimulated⁴⁵Ca uptake, although the characteristic patterns of differentiation (extension of neurite-like processes, etc.) and that of effect by Ca agonist or antagonists on NG108-15 cells and PC12h cells were similar.     

Specific protein phosphorylation during cyclic AMP-mediated morphological reversion of transformed cells

Treatment of transformed Chinese hamster ovary cells with dibutyryl cAMP or other agents that elevate cAMP results in the acquisition of growth and morphology characteristic of normal fibroblasts. The role of specific protein phosphorylation in this process of morphological reversion has been examined using metabolic labelling of Chinese hamster ovary (CHO) cells with ³²P-orthophosphate in the presence or absence of N⁶O^2′-dibutyryladenosine 3′:5′-cyclic monophosphoric acid (Bt₂cAMP). Analysis of labelled cultures by SDS gel electrophoresis and radioautography demonstrate dramatic changes in the phosphorylation of only 2 cellular proteins during reverse transformation. A 55,000 dalton protein (pp55) was phosphorylated and a 20,000 dalton protein (pp20) was dephosphorylated. The time course of these events was consistent with the kinetics of morphological reversion. The lower molecular weight species, pp20, was dephosphorylated within 15–30 minutes, prior to all morphological changes except membrane tranquilization. The higher molecular weight protein, pp55, was maximally phosphorylated over 1–2 hours following addition of Bt₂cAMP, paralleling early stages in the establishment of fibroblastic form. The phosphorylated forms of pp20 and pp55 were both extracted from cellular cytoskeletons by 0.5% Triton X-100, but analysis of ³⁵S-methioninelabelled cultures suggested that unphosphorylated pp 20 may be bound to the cytoskeleton. Since pp20 was found to comigrate with the 20,000 dalton myosin light chain, it is possible that dephosphorylation of CHO cell myosin induced by cAMP may alter its interaction with actin microfilaments and modulate the assembly of stress fibers during morphological reversion.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G₁ phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G₁–S boundary) of G₁ phase. The kinetics of exit from G₁ phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G₁ beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Existence of a commitment program for mitosis in early G1 in tumour cells

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G₁ (which we have designated G₁ pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G₁ pm and enter G₀. The G₁ pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G₁ or pre S phase (G₁ ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82 , 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [³H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G₁ (3-4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G₁ amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G₁ pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G₁ pm-like stage. Our conclusion is that G₁ pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

X-ray Sensitivity of HeLa S3 Cells in the G2 Phase: Comparison of Two Methods of Synchronization

The sensitivity of HeLa S3 cells to 220 kv X-rays was measured in terms of cell survival (colony development) during the G2 phase of the cell generation cycle, employing two procedures designed to free G2 cultures from contaminating cells from other phases of the cycle. Treatment of synchronous cultures (obtained initially by mitotic selection) with high specific activity tritiated thymidine (HSA-³HTdR) selectively eliminated S phase cells, while addition of vinblastine permitted removal of cells as they entered mitosis. It was found that HeLa S3 cells become increasingly sensitive as they progress through G2. The pattern of sensitivity fluctuations observed in synchronous HeLa S3 populations selected by the foregoing method was compared with that found in synchronous cultures prepared by the HSA-³HTdR method of Whitmore. The latter method had been used previously with mouse L cells, which were found to undergo a different pattern of sensitivity fluctuations. The two methods yield similar results for HeLa cells in the S and G2 phases of the cycle. It may be concluded, therefore, that the discrepancies between HeLa and mouse L cells do not arise from methodological factors, but represent fundamental differences between the cell types.     

Autoradiographic study of the effect of 5-aminouracil on the S phase and mitosis of barley root meristems

The effect of 5-aminouracil on the S phase and mitosis in root meristems of barley embryos cultivated in the liquid nutrient solution was followed. Embryos were cultivated in different concentrations of 5-aminouracil (200 ppm, 400 ppm and 750 ppm) for 48 h. The drug postponed the onset of mitosis. In the lowest concentration used, synchronization was observed even in the presence of 5-aminouracil. In higher concentrations, mitosis was suppressed irregularly with increasing concentration. 5-aminouracil slowed down the rate of DNA synthesis during S phase and prolonged the S phase, as measured by the utilization of [³H] thymidine. The drug does not influence considerably the entry of cells into the S phase. The transition from G₂ to mitosis is delayed in the presence of 5-aminouracil, especially in higher concentrations. After prolonged treatment with 5-aminouracil, all the effects of the drug on the mitotic cycle decrease continuously.     

Arrest of cell division blocks the utilization of polyamines in synchronized cultures of photoautotrophically grown Euglena

A trimodal change in the cellular levels of three major polyamines: spermidine, N,N′-bis(3-aminopropyl)-1, 3-propanediamine (BAP) and 3,3′-diaminodipropylamine (DAD) was observed during two successive cell cycles in synchronously dividing cultures of the algal flagellate, Euglena gracilis Z photoautotrophically grown in a 24-h light-dark cycle. The intracellular levels of these three polyamines decreased as cells divided and then were enhanced as cells exited the G₁ phase and proceeded through the S and G₂ phases. Spermidine, BAP and DAD concentrations increased about 2.5-fold during the S phase. Putrescine and 1,3-diaminopropane levels did not vary significantly. One peak of polyamine synthesis occurred in the G₁ phase prior to DNA synthesis, followed by a second more important peak during the S-G₂ phases before cell division; both peaks were observed during the light period. A third minor peak was observed during the pre-G₁ (or G₀) phase in the dark period after mitosis had been completed. In contrast, when the cells attained the “stationary” phase of growth, there was no significant increase in the content of polyamines during the light period although spermidine and BAP increased slightly twice during the dark period (putrescine and 1,3-diaminopropane and DAD levels remained almost constant). To ascertain whether the synthesis of polyamines was merely a direct effect of the photoperiod, parallel experiments with synchronous cultures were carried out in the presence and absence of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, a photosynthetic inhibitor. Although a slight decrease in the concentration of polyamines was observed, the three maxima of polyamines synthesis were observed as in normal cultures. These results clearly suggest that polyamine biosynthesis is closely related to DNA replication and cell division in Euglena cells.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [³H](+)PN200-110 (PN: L-type Ca channel antagonist) and [¹²⁵I]-conotoxin G VI A (-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt₂cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated⁴⁵Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt₂cAMP (unpublished results). The specific binding of [³H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [³H](+)PN. Scatchard analysis showed that the specific binding of [³H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt₂cAMP, but that the apparent Kd value for the specific binding of [³H](+)PN was not influenced by treatment with Bt₂cAMP. The specific binding of [³H](+)PN to crude membranes from Bt₂cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(–)PNBay K 8644diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [³H(+)PN. On the other hand, [¹²⁵I]-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated⁴⁵Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [¹²⁵I]-CgTX on Bt₂cAMP-treated or untreated NG108-15 cells.     

Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [³⁵S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G₂ phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G₂ phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G₂ phase. In the G₁ and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)⁺ RNA isolated at each phase. Three poly(A)⁺ RNAs increased in amount from the G₁ to the S phase and one poly (A)⁺ RNA increased preferentially from the G₂ phase to cytokinesis.     

Effect of cyclic amp on suncus murinus liver serine-aminotransferase and serine dehydratase

• 1.1. Suncus murinus was injected dibutyryl adenosine 3′,5′-cyclic monophosphate (Bt₂cAMP) and assayed serine-glyoxylate aminotransferase (EC 2.6.1.45) and serine dehydratase (EC 4.2.1.13).
• 2.2. Serine dehydratase was induced 4-fold by Bt₂cAMP. The K_m values of the induced enzyme for l-serine and pyridoxal 5′-phosphate was 57 mM and 3.0 μM, respectively. The enzyme had a pH optimum at pH 10.0. These kinetic properties and pH optimum were same as those of the enzyme from the control. Both the holoenzyme and the apoenzyme increased to the same extent by Bt₂cAMP.
• 3.3. Serine-glyoxyate aminotransferase activity was decreased slightly by the Bt₂cAMP injection. The holoenzyme activity was increased, but the apoenzyme decreased. K_m values for l-serine and glyoxylate of this enzyme were 6mM and 0.2 mM, respectively, without change by Bt₂cAMP.