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PubMed1986

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Activation of adenylate cyclase in human platelet membranes by guanosine 5'-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

Incubation of platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate causes a slow increase in GS (stimulatory GTP-binding protein) activation of adenylate cyclase. Mg2+ is necessary for this slow activation. This process is inhibited in the presence of ATP, and inhibition is greater if cyclic AMP is also included in the incubation. Adenosine 5'-[beta gamma-imido]triphosphate instead of ATP in the incubation facilitates the slow activation in the presence of cyclic AMP, and incubation of membranes with cyclic-AMP-dependent protein kinase inhibitor decreased inhibition of the slow activation of adenylate cyclase by ATP and cyclic AMP. A protein of 45 kDa in platelet membranes is phosphorylated in a cyclic-AMP-dependent manner. The transition from a reversibly activated form of GS to an irreversibly activated form is substantially slower in the presence of ATP and cyclic AMP. We propose that guanosine 5'-[beta gamma-imido]triphosphate-activated GS may exist in phosphorylated or non-phosphorylated forms, and that the non-phosphorylated form is the more active of the two species. The non-phosphorylated form of GS may correspond to the irreversibly activated state.

Adenosine Triphosphate↗
PubMed1987

Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.

Adenylyl Cyclases↗
PubMed1987

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.

Animals↗
PubMed1987

Selective regulation of the amount of catalytic subunit of cyclic AMP-dependent protein kinases during isoprenaline-induced growth of the rat parotid gland.

Stimulation of growth of the rat parotid gland by repeated injection of the beta-agonist isoprenaline led to a significant decrease in the activity of cyclic AMP-dependent protein kinases. Immunochemical quantification of the catalytic (C) and regulatory (RI and RII) subunits of the cyclic AMP-dependent protein kinases type I and type II revealed a loss of 65% of the immunochemically measurable amount of catalytic subunit C. The amount of the regulatory subunits, however, remained constant. The observed decrease in C-subunit was not due to a translocation of the molecule to cellular membranes or to an inhibiting effect of the heat-stable inhibitor of cyclic AMP-dependent protein kinases. A selective decrease in only the C-subunit was also observed after a brief exposure to isoprenaline leading to the stimulation of DNA synthesis. Under these conditions, the decrease was observed at the onset of DNA synthesis (17 h after injection), but not at the the time of an earlier small cyclic AMP peak (13 h after injection) or at the time of maximal DNA synthesis (24 h after injection). The results indicate that the amount of the catalytic subunit of cyclic AMP-dependent protein kinases can be regulated independently from that of the regulatory subunits. The time-limited occurrence of the specific change in the amount of the C-subunit suggests that such a regulation is of physiological significance and that it may participate in cyclic AMP-mediated events involved in the control of cellular proliferation.

Animals↗
PubMed1988

Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme.

AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl ('adult' isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl ('fetal' isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The 'fetal' isoform had about 5-fold higher half-saturation constant (S0.5) value than the 'adult' form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The 'adult' isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the 'fetal' isoform. In the muscle of 16-week-old human fetus, the 'fetal' isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase.

AMP Deaminase↗
PubMed1992

Retinoic acid stimulates expression of thrombomodulin, a cell surface anticoagulant glycoprotein, on human endothelial cells. Differences between up-regulation of thrombomodulin by retinoic acid and cyclic AMP.

Thrombomodulin (TM) is a surface protein on endothelial cells, and represents one of the most valuable regulatory factors in the anticoagulant system. In this paper, we demonstrate that retinoic acid (RA) causes an increase in TM antigen on human umbilical vein endothelial cells (HUVECs) in vitro. The effect of RA on the surface TM level of HUVECs was dose-dependent in the range from 0.01 to 10 microM-RA. Antigen levels began to increase 3 h after addition of 10 microM-RA, and plateaued at a maximum level of approx. 2.5 times that of the untreated control at 24 h. TM levels remained at a maximum for a further 12 h, and then gradually decreased. The effects of RA on cell surface TM activity and antigen levels were parallel in all experiments. TM expression was also increased by treatment with 10 microM-retinal or 10 microM-retinol for 24 h, though the increases were approx. 70% and 30% respectively of that produced by 10 microM-RA. Pretreatment of HUVECs with cycloheximide inhibited the effect of RA. When HUVECs were incubated with both 10 microM-RA and 5 mM-8-bromo cyclic AMP (or 1 mM-3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor), the increase in TM antigen was greater than that observed with either compound alone. Northern blot analysis showed that treatment of HUVECs with 8-bromo cyclic AMP, RA or RA plus 8-bromo cyclic AMP increased TM mRNA levels by 2.2-, 4.5- and 5.5-fold respectively compared with the untreated control. Furthermore, no significant difference in cellular cyclic AMP levels was observed between RA-treated and control cells. These results indicate that the expression of TM is not only controlled by the intracellular cyclic AMP level but is also affected by RA, and suggest that RA-induced up-regulation of TM on HUVECs is independent of cyclic AMP regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

The effects of elevated cyclic AMP levels on histamine-H1-receptor-stimulated inositol phospholipid hydrolysis and calcium mobilization in the smooth-muscle cell line DDT1MF-2.

The effects of raising cyclic AMP levels, by forskolin stimulation, beta-adrenoceptor activation or cyclic AMP phosphodiesterase inhibition, on inositol phospholipid hydrolysis and increases in intracellular free [Ca2+] ([Ca2+]i) elicited by a range of agonists have been investigated in the hamster vas deferens smooth-muscle cell line DDT1MF-2. Isoprenaline (log [EC50 (M)] = -7.7 +/- 0.2), forskolin and the type IV cyclic AMP phosphodiesterase inhibitor rolipram elicited significant increases in the accumulation of cyclic [3H]AMP. Pretreatment with forskolin (10 microM) attenuated histamine (100 microM)- and N6-cyclopentyladenosine (CPA; 300 nM)-induced release of intracellular Ca2+, observed when cells are stimulated in Ca(2+)-free buffer containing 0.1 mM EGTA. Forskolin had no effect on ATP (100 microM)- or bradykinin (1 microM)-stimulated release of intracellular Ca2+. Histamine-induced intracellular Ca2+ release was also inhibited by pretreatment with rolipram (100 microM) or the membrane-permeant cyclic AMP analogue (Sp)-adenosine 3',5'-monophosphothioate (100 microM). Isoprenaline (1 microM) pretreatment (in the presence of 10 microM rolipram, a concentration which on its own did not decrease the histamine response) attenuated histamine-induced intracellular Ca2+ release. Forskolin inhibited histamine (100 microM)- and CPA (100 nM) stimulated accumulation of [3H]-inositol phosphates, but was without effect on ATP or bradykinin responses. Addition of forskolin (in the presence of 100 microM rolipram) after the cells had been stimulated with histamine (in experiments initiated in Ca(2+)-free buffer) inhibited the rise in [Ca2+]i observed when extracellular Ca2+ (2 mM) was re-applied (owing to receptor-mediated Ca2+ influx). Finally, the refilling of intracellular Ca2+ stores (after receptor-mediated Ca2+ influx is blocked by mepyramine) can be demonstrated in the presence of raised cyclic AMP levels.

Animals↗
PubMed1994

Protein kinase C-dependent cyclic AMP formation in airway smooth muscle: the role of type II adenylate cyclase and the blockade of extracellular-signal-regulated kinase-2 (ERK-2) activation.

Bradykinin activates adenylate cyclase via a pathway that involves the 'up-stream' regulation of phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine and activation of protein kinase C (PKC) in airway smooth muscle [Stevens, Pyne, Grady and Pyne (1994) Biochem. J. 297, 233-239]. Coincident signal (Gs alpha and PKC) amplification of the cyclic AMP response can be completely attenuated either by diverting PLD-derived phosphatidate or by inhibiting PKC. In this regard, the coincident signal detector type II adenylate cyclase is expressed as a 110/112 kDa polypeptide in these cells. PKC alpha is not involved in the activation of adenylate cyclase, since a B2-receptor antagonist (NPC567, 10 microM) blocked its bradykinin-stimulated translocation to the membrane and was without effect against both bradykinin-stimulated PLD activity and cyclic AMP formation. Cyclic AMP formation can also be activated by platelet-derived growth factor (PDGF), via a PKC-dependent pathway, although the magnitude of the response is less than that elicited by bradykinin. Nevertheless, these results indicate that multiple receptor types employ PKC to initiate cyclic AMP signals. PDGF (10 ng/ml) elicited the marked sustained activation of extracellular-signal-regulated kinase-2 (ERK-2), whereas bradykinin (1 microM) provoked only modest transient activation of ERK-2. Deoxyadenosine (0.1 mM), a P-site inhibitor of adenylate cyclase, blocked bradykinin-stimulated cyclic AMP formation and converted the activation of ERK-2 into a sustained response. Thus the PKC-stimulated cyclic AMP response can limit the activation of ERK-2 in response to bradykinin. These studies indicate that the integration of distinct signal pathways by adenylate cyclase can determine the kinetics of ERK activation, an enzyme that appears to be important for mitogenic progression.

Adenylyl Cyclases↗
PubMed1995

Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with respect to the cell signal regulation of mitogenesis in airway smooth muscle.

Adenylyl Cyclase Inhibitors↗
PubMed1996

The differential regulation of cyclic AMP by sphingomyelin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in airway smooth muscle.

We report that sphingosine and short-chain ceramides activate adenylate cyclase and stimulate intracellular cyclic AMP formation in airway-smooth-muscle (ASM) cells. In each case, there is a conditional requirement for GTP-Gs alpha. Sphingosine utilizes a protein kinase C-dependent pathway to elicit activation of adenylate cyclase, whereas for short-chain ceramides the mechanism remains unidentified. In contrast, sphingosine phosphate inhibits Gs-stimulated cyclic AMP formation via a Gi-dependent mechanism. Therefore, the potential interconversion of sphingosine and sphingosine phosphate is a switch that can elicit reciprocal changes in cyclic AMP levels. This may have a significant impact upon the regulation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal specific kinase (JNK) by sphingolipids and may help to explain how growth factors that utilize these second messengers evoke pleiotropic responses such as proliferation and cell survival. In this context, short-chain ceramides are poor stimulators of ERKs in ASM cells, and sphingosine is inactive, whereas both sphingolipids are powerful activators of the JNK module. Activated JNK catalyses N-terminal phosphorylation of c-Jun, a kinase cascade that programmes growth arrest. Therefore, in blocking ceramide-stimulated ERK-2 activity, cyclic AMP may allow the ceramide-dependent activation of JNK to programme cells to opt out of the cell cycle. In contrast, sphingosine phosphate activates ERK-2, potentiates growth-factor-stimulated DNA synthesis and fails to activate JNK, indicating that its sequential formation from ceramide and sphingosine may commit cells to DNA synthesis. ERK-2 can be activated by both cyclic AMP-sensitive c-Raf-1 kinase-dependent and cyclic AMP-insensitive c-Raf-1 kinase-independent pathways in ASM cells. In this context, sphingosine phosphate activates ERK-2 exclusively via c-Raf-1 kinase. Sphingosine phosphate-stimulated ERK-2 activity is also abolished by pertussis toxin, indicating that c-Raf-1 kinase is activated via a Gi-dependent mechanism.

Adenylate Cyclase Toxin↗
PubMed1992

Cyclic AMP and the regulation of cholesterol metabolism.

Cyclic AMP has been implicated to a greater or lesser extent in the regulation of four key enzymes which interact to regulate intracellular cholesterol metabolism; HMG CoA reductase; ACAT; cholesteryl ester hydrolase; and cholesterol 7 alpha hydroxylase. The relationship between these enzymes and the sites where current evidence suggests that cyclic AMP may be involved are summarized in Fig. 3. Cholesterol 7 alpha hydroxylase controls the catabolism of cholesterol to bile acids in the liver, and thus its removal from the body via the bile, but does not have a major role in cholesterol metabolism in extrahepatic tissues. It is clear that cyclic AMP is able to influence the activity of this enzyme in liver sub-cellular fractions and isolated hepatocytes in vitro, and studies in our laboratory have shown that changes in Ca2+ fluxes within the cell may be important in its mechanism of action. Whether or not the cyclic nucleotide has a role regulating cholesterol 7 alpha hydroxylase activity in vivo, however, is not known. HMG CoA reductase is inactivated by phosphorylation both in vitro and in vivo, but although cyclic AMP and glucagon have been shown to inhibit the enzyme, cyclic AMP-dependent protein kinase is not directly involved. The exact mechanism by which the cyclic nucleotide influences the system remains unclear, but it may be related to activation of microsomal phosphatases. The activity of ACAT has been shown to be modulated by phosphorylation in a number of tissues in vitro, but the involvement of cyclic AMP has not been unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Elevated arterial cyclic AMP levels during the onset of renal hypertension in rats.

1. To determine the possible role of arterial cyclic AMP in the pathogenesis of hypertensive vascular hypertrophy and hyperplasia, the changes in the level of this nucleotide were studied during the development of renal hypertension in rats with aortic ligation between the renal arteries. 2. A twofold increase in the cyclic AMP level of the thoracic aorta was observed in 9-day hypertensive rats when compared with sham-operated controls. At this time the total amounts of DNA and collagen were unchanged, although a marked increase in arterial fibrous protein was already present. 3. Arterial cyclic AMP remained significantly elevated in the thoracic aorta of 30-day hypertensive animals. At this time the hypertensive vascular alterations had reached completion as shown by the abnormal accumulation of collagen, DNA and non-fibrous protein. 4. Contrary to the events taking place in the thoracic aorta, a marked decrease in cyclic AMP was present in the abdominal portion, which was protected from high blood pressure by the aortic ligature. In this segment decreased cyclic AMP coexisted with an unchanged collagen content and a diminution in the contents of DNA and non-fibrous protein. 5. Thus a marked increase in arterial cyclic AMP precedes the initiation of DNA replication and collagen accumulation in vascular territories subjected to high blood pressure. These studies suggest the participation of this nucleotide in the vascular growth induced by hypertension.

Animals↗
PubMed1994

In vivo evidence that nonneuronal beta-adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum.

We applied in vivo microdialysis to assess the effects of dopaminergic and beta-adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1 mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D1 agonist SKF 38393 and D2 antagonist sulpiride produced modest increases. It is interesting that the beta-adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the beta-adrenoceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1 mM), by a 5-h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neurotoxin, kainic acid (2.5 micrograms), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate the beta-adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and beta-adrenoceptor-stimulated cyclic AMP efflux are substantially dependent on intact glial cells.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1997

Two intracellular signaling pathways for the dopamine D3 receptor: opposite and synergistic interactions with cyclic AMP.

As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108-15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50-75% the quinpirole-induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6-16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of > 1 microM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.

Cell Line↗
PubMed1997

Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP.

Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either AA or DHAA. AA uptake was Na+-dependent and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), whereas DHAA uptake was Na+-independent and DIDS-insensitive. DHAA uptake was inhibited by cytochalasin B, D-glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2-deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.

Animals↗
PubMed1998

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers.

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.

1-Methyl-3-isobutylxanthine↗
PubMed1983

The stimulus-secretion coupling of glucose-induced insulin release. LIII. Calcium-dependency of the cyclic AMP response to nutrient secretagogues.

Above a threshold value in excess of 5.6 mM, D-glucose increases the amount of cyclic AMP measured by radioimmunoassay in pancreatic rat islets and their surrounding incubation medium. As judged from the cyclic AMP content of islets exposed to isobutylmethylxanthine (1.0 mM), the glucose-induced increment in the rate of cyclic AMP generation represents a rapid and sustained phenomenon. The stimulant action of glucose on cyclic AMP accumulation is mimicked by L-leucine, and L-glutamine, these amino acids acting synergistically of one another. Trifluoperazine slightly decreases but fails to abolish the effect of glucose. In the absence of extracellular Ca2+, however, the cyclic AMP response to D-glucose, L-leucine and/or L-glutamine is severely impaired. These findings are compatible with the view that an increase in the generation rate of cyclic AMP participates in the process of nutrient-stimulated insulin release. This increase could be secondary to the nutrient-induced accumulation of Ca2+ in the islet cells leading to activation of adenylate cyclase by calmodulin.

1-Methyl-3-isobutylxanthine↗