Cyclic AMP Inhibits Activation of Mitogen-Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes

Abstract: The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 m M and isoproterenol at 10 µ M inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with ³²P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.     

Induction of DNA synthesis by dichloroisoproterenol without initial rise of the cAMP level in the parotid gland of mouse

The increase in the cAMP concentration in the parotid gland of mouse, observed shortly after administration of isoproterenol, was not detected after dichloroisoproterenol, a β-adrenergic blocking agent. The latter compound, however, could induce DNA synthesis which was comparable to that induced by IPR in several respects. The results strongly suggest that the initial rise of cAMP concentration in the parotid gland after IPR injection is not related directly to the initiation of the stimulated DNA synthesis.     

cAMP and sodium transport in the freshwater crayfish, Cherax destructor.

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na(+)-K(+)-ATPase activity in gill tissue. The concentration of cAMP and activity of Na(+)-K(+)-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol x l(-1) Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na(+)-K(+)-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na(+)-K(+)-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1-2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3-6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP-independent pathway in osteoblastic clone MC3T3-E1 cells

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.     

急性压力超负荷后心肌cAMP与肾素血管紧张素系统活化的关系

为阐明急性压力超负荷后心肌细胞内ｃＡＭＰ浓度升高和心肌肾素血管紧张素系统活化之间是否存在内在因果联系,用腹主动脉缩窄的方法建立急性压力超负荷大鼠模型。发现在术后１ｈ心肌组织中血管紧张素转换酶（ＡＣＥ）ｍＲＮＡ及蛋白表达均显著增加,ＡＣＥ活性及血管紧张素Ⅱ（ＡｎｇⅡ）含量也明显升高,并在高水平。同时,心肌组织ｃＡＭＰ含量于术后０．５ｈ明显增加,术后５ｄ时达峰值,术后３０ｄ降至正常。在心肌细胞培养的基     

cAMP and sodium transport in the freshwater crayfish, Cherax destructor

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na⁺-K⁺-ATPase activity in gill tissue. The concentration of cAMP and activity of Na⁺-K⁺-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol.l⁻¹ Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na⁺-K⁺-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na⁺-K⁺-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1–2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3–6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.     

Calcium thresholds in the activation of DNA and RNA synthesis in cultured planarian cells: relationship with hormonal and DB cAMP effects

DNA and RNA syntheses were reduced to a basal level in dissociated planarian cells grown for 48 h in a Ca2+-free medium. These syntheses could be triggered anew by raising the Ca2+ concentration in the medium. Serotonin could be substituted for Ca2+ in stimulating DNA synthesis by Ca2+-depleted cells, while dopamine greatly enhanced RNA synthesis in these cells. When Ca2+ concentration was raised in hormone-treated cultures, DNA synthesis was again slightly increased but RNA synthesis was depressed. Both hormonal effects were completely inhibited by the anticalmodulin drug trifluoperazine. As serotonin and dopamine are both known to stimulate the adenylate cyclase system, it was further investigated whether the hormonal effects were mediated by cAMP. Indeed, a DB cAMP concentration of 1 microM increased DNA labelling when applied for 8 h to Ca2+-depleted cultures. However, when Ca2+ was present, the 8-h treatment with 1 microM DB cAMP was inhibiting. A 4-h pulse with 1 microM DB cAMP just after Ca2+ addition was a condition for a high stimulation of DNA labelling. The other DB cAMP concentrations used, 0.1 and 10 microM, reduced DNA labelling. In the absence of Ca2+, RNA labelling was only slightly increased by 0.1 microM DB cAMP, but was highly stimulated by a 4-h treatment of 1 microM DB cAMP in the presence of Ca2+. The noted effects with 1 or 0.1 microM DB cAMP on DNA or RNA labelling corresponded to true changes in synthesis rather than alterations of the specific activity of the nucleotide pool by DB cAMP. Besides, it was precluded that these effects were due to butyrate issued from DB cAMP degradation. It was further shown that DB cAMP at 1 microM increased Ca2+ uptake in planarian cells, whereas the other concentration reduced it. This observation might explain the stimulating effect on nucleic acid synthesis of 1 microM DB cAMP applied at the appropriate moment. Based on these results it seems that, for triggering RNA synthesis, the threshold value of Ca2+ was lower than for DNA synthesis. These Ca2+ thresholds might be reached, in the absence of Ca2+ in the medium, by treatments with DB cAMP or hormones at the appropriate doses and periods. This interpretation is in agreement with the succession of biochemical events described in regenerating planarians and suggests that these events might be causally related.     

Effect of serum from chickens treated with clenbuterol on myosin accumulation, β-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 microM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The beta-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 microM isoproterenol, limited increases of 12-20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 microM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher betaAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Adenosine 3'':5''-monophosphate content and actions in the division cycle of synchronized HeLa cells

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.     

Aurintricarboxylic acid (ATA) and DNA synthesis. I. Inhibition of DNA synthesis by ATA in Go cells stimulated to proliferate.

Aurintricarboxylic acid (ATA) at a concentration which produces 40% inhibition of protein synthesis, inhibits completely isoproterenol-stimulated DNA synthesis in mouse parotid glands. The drug was found to interfere with some essential changes occurring during the prereplicative phase of IPR-stimulated DNA synthesis. It inhibits the increase in ribosonal protein synthesis that takes place by 2 h after stimulation. The peak of ribosonal RNA that occurs 8 h after isoproterenol was also abolished by ATA. Since the drug completely inhibits isoproterenol-stimulated DNA synthesis, these results suggest that the control of ribosome production may be involved in cell growth activation. In view of the finding that ATA first inerferes with the binding of adenylate-rich RNA to polysomes, it was suggested that the drug may act by preferentially inhibiting that fraction of protein synthesis dependent on the newly transcribed messenger RNA.     

Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Mitogenic stimulation in isoproterenol treated cells of dictyostelium discoideum.

Amoebae of the cellular slime mould Dictyostelium discoideum showed stimulated mitogenic activity when exposed to 200 microM isoproterenol, an activator of adenyl cyclase, for 30 min. Approximately 40% increase in cell proliferation was found at 48 h after isoproterenol treatment. A faster and larger plaque formation as well as higher uptake of FITC-labelled E. coli indicates greater phagocytotic activity in the treated cells. A concurrent increase in DNA and protein syntheses was also recorded in the treated cells. Administration of 400 microM caffeine or 200 microM (+) propranolol brought down the isoproterenol-induced elevation in the cell division rate to control levels. These results are discussed in relation to a precocious activation of adenyl cyclase in the treated cells leading to a transient but significant increase in cell division in this organism.     

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.     

The labile nature of the insulin signal(s) for the stimulation of DNA synthesis in mouse lens epithelial and 3T3 cells

A kinetic study was carried out to assess the stability of the intracellular signal(s) generated by insulin in quiescent cells for the stimulation of DNA synthesis. Using murine lens epithelial cells and Swiss 3T3 cells in culture, it was found that insulin stimulated DNA synthesis after a lag of 14.5 h. If, however, 6 h after the addition of insulin to the cells, the insulin-containing media were totally removed, followed by the addition of fresh media (even if insulin was returned to the medium within approximately 10 min), a 14.5-h lag still remained after insulin readdition before DNA synthesis started. In another set of experiments, the insulin was removed after 6 h by diluting its concentration approximately 60,000-fold. In this case, if insulin was at the diluted concentration for approximately 60 min before being added back, a full 14.5 h was necessary for the start of DNA synthesis. The half-time for loss of signal was 2 +/- 1 min for total washout and 18.4 +/- 0.5 min for the dilution experiment. These results indicate that the intracellular signal(s) for DNA synthesis produced by the binding of insulin to its cellular receptor are extremely transitory in nature. The signal disappears at approximately the same rate that insulin dissociates from the receptor. Thus, insulin must be constantly binding to the membrane receptor in order to keep the key signal(s) at a high enough level for the cell to progress on to S phase. Early events, such as specific protein synthesis, changes in ion flux, changes in cellular metabolism, and changes in cellular pH, may be essential, but they are not sufficient to cause a cell to progress on to S phase. Addition of sodium vanadate to the cell is found to stabilize the messenger such that there is no loss of signal when insulin is removed. These data are consistent with the tyrosine-phosphorylated insulin receptor or a product of its action being the signal.     

Prereplicative changes in the soluble calmodulin of isoproterenol-activated rat parotid glands

Cells of rat parotid glands were maximally stimulated to initiate DNA synthesis by injecting into the animal a single dose of 25 to 150 mg of isoproterenol/ kg of body weight. During the 18- to 21-hr prereplicative period following injection of the highest dose of the drug, there were two predominant and transient redistributions of calmodulin from the bound to the soluble form, which tripled the level of soluble calmodulin at 3 hr and again at 18 hr just before the initiation of DNA synthesis. A small (50%) increase in total calmodulin was observed only during the early (3-h) prereplicative surge of soluble calmodulin. The late, pre-DNA-synthetic surge of soluble camodulin and the initiation of DNA synthesis were both prevented in rats that lacked their parathyroid-thyroid gland complex and had been hypocalcemic for 48 or 72 hr. Unlike the effect of high doses of isoproterenol, low doses (e.g., 25 mg/kg body weight) of the β-adrenergic drug could maximally stimulate DNA synthetic activity without the later pre-DNA-synthetic surge of soluble calmodulin, suggesting that any apparent correlation between the level of calmodulin and DNA synthesis may be spurious and that an actual increase in the level of soluble calmodulin just before the onset of DNA synthesis was not a prerequisite for DNA synthetic activity in parotid cells.     

In vivo changes in free choline level induced by autonomic agonists in mouse organs, including three major salivary glands

Whether free choline levels are changeable in vivo in response to different types of autonomic agonists was examined in several mouse organs. Upon one subcutaneous injection of isoproterenol, phenylephrine and pilocarpine, choline levels in whole organ decreased, increased and decreased, respectively, in various organs within 30 min and returned to initial levels in a day. In the three major salivary glands, a delayed choline elevation also appeared on day 2 after one isoproterenol injection and subsided by day 6. Only in the three salivary glands more choline was accumulated after 10 once-a-day injections of isoproterenol than after one isoproterenol injection. Neither phenylephrine nor pilocarpine induced comparable choline accumulation in any organs examined. Isoproterenol injection repeated at a 2-day interval augmented the subsequent, delayed choline elevation. Examination with dobutamine and the adenylyl cyclase activator 6-(3-dimethylaminopropionyl)forskolin suggested that isoproterenol-induced immediate choline lowering was down-stream of cAMP synthesis and linked to cAMP more tightly than the choline accumulation, though both choline changes occurred via beta1-adrenergic receptors. Choline levels in the salivary glands also changed depending on the form of diet given and particularly in the parotid gland in parallel with gland weights. These results provide the first evidence for the autonomic control of intracellular choline levels; intracellular choline levels might be an integral part of the autonomic signalling pathway.