Effects Of Elevated cAMP Research Articles

Elevated cAMP gives short‐term inhibition and long‐term stimulation of hepatocyte DNA replication: Roles of the cAMP‐dependent protein kinase subunits

The study reports the role of the isozyme forms (cA-PKI and cA-PKII) and subunits (R and C) of cAMP-dependent protein kinase in mediating the acute depression of hepatocyte DNA replication by elevated cAMP. Combinations of cAMP analogs preferentially activating cA-PKI or II showed that either isozyme could inhibit DNA replication. The effects of glucagon and cAMP analogs were counteracted by the cAMP antagonist RpcAMPS, implicating the necessity for cA-PK dissociation in cAMP action. The effect of elevated cAMP was mimicked by microinjected C subunit, but not by the RI subunit of cA-PK. Hepatocytes under continuous cAMP challenge more than regained their replicative activity. This tardive stimulatory effect of cAMP was enhanced by insulin and blocked by dexamethasone, and was preceded by downregulation of cA-PK. In conclusion, a burst of cAMP acutely inhibits hepatocyte G1/S transition in late G1 regardless of hormonal state. In the presence of high glucocorticoid/low insulin the inhibition persists. At high insulin/low glucocorticoid the inhibitory phase is followed by a prolonged stimulation of DNA replication. Downregulation of endogenous cA-PK is a mechanism for escape from the inhibitory action of highly elevated cAMP.

Anti-class II antibodies potentiate IgG2a production by lipopolysaccharide-stimulated B lymphocytes treated with prostaglandin E2 and IFN-gamma.

IFN-gamma secretion by Th1 cells has been shown to preferentially promote the production of IgG2a in LPS-stimulated murine B lymphocytes. We recently reported that PGE2 potentiated the ability of IFN-gamma to augment IgG2a production in both Ag-specific and polyclonal systems via a cAMP-dependent pathway. Because antibodies (Ab) directed against class II MHC molecules have been shown to induce a rise in B cell cAMP, we hypothesized that this event, like PGE2 treatment, would promote the production of IgG2a. In this manuscript, cultures of small and large B cells treated with anti-Ia Ab are shown to produce significantly higher levels of IgG2a, compared with cultures treated only with IFN-gamma and LPS. Moreover, the combined treatment of B lymphocytes with IFN-gamma and PGE2 followed by anti-Ia and LPS resulted in a fourfold rise in IgG2a levels compared with IFN-gamma and LPS. Only anti-class II, but not anti-class I Ab, stimulated IgG2a production. Utilizing an ELISA spot assay, the frequency of IgG2a-secreting B cells was determined to be elevated fourfold in anti-Ia treated B cells. B cell cultures incubated with either PGE2 or anti-Ia exhibited elevated levels of cAMP and treatment with IFN-gamma primed these lymphocytes to the cAMP-elevating effects of either PGE2 or anti-Ia. Finally, RpcAMP, a cAMP antagonist that blocks cAMP from activating protein kinase A, prevented the increased production of IgG2a induced by anti-Ia Ab. These results support the theory that a cAMP pathway exists that promotes B cell IgG2a production. Within this pathway, IFN-gamma sensitizes B lymphocytes to cAMP elevators such as anti-class II Ab, and in conjunction with LPS, causes an increase in the frequency of IgG2a-secreting cells and the amount of IgG2a produced. These observations suggest that, after exposure to viral Ag in vivo, interaction between IFN-gamma-primed murine B cells and T cells will potentiate production of IgG2a, the predominant murine anti-viral Ig.

Embryonic cAMP and developmental potential in Drosophila melanogaster.

Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action.

Regulation of mesangial cell adhesion and shape by thrombin.

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS)

The hepatocyte calcium oscillator

Hepatocytes stimulated with calcium-mobilising agonists generate free Ca transients whose frequency is modulated by hormone concentration. Importantly, the time-course of individual free Ca transients is independent of agonist dose but does change with agonist species. A receptor-controlled model in which protein kinase C provides negative feedback directed against the different receptors, or receptor-specific G proteins, has been proposed in order to explain the agonist-specificity of the falling phase of the free Ca spikes. Here we show further evidence, from mixing of hormones and from the effects of elevated cAMP, of receptor-specific information within the spikes.

Roles of actin filaments and three second-messenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth.

In a previous study (J. Cell Biol. 109: 1229-1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin-stabilizing drug phalloidin could block or reverse calcium-ionophore-induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium-induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second-messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium-permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2-AM-measured calcium levels indicated that the effects of cAMP manipulations could be partially explained by a cAMP-induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent mechanism.

Concentration-dependent effect of met-enkephalin on human polymorphonuclear leukocytes.

Met-enkephalin in 10(-9)-10(-7)M concentrations exerts an ADCC-stimulating effect on human PMNLs through naloxone-sensitive opiate receptors, elevating the cytoplasmic free Ca2+ and cGMP levels. In higher, 10(-6)-10(-5)M concentrations ME has a cAMP-elevating effect and a rapid 45Ca2+ influx was observed. This latter effect of ME, which is also associated with the suppression of ADCC activity, was abolished by the enkephalinase inhibitor, puromycin. A strong relationship is suggested between the suppressing effect of metenkephalin, enkaphalinase and the protein kinase C system.

Effects of forskolin on spontaneous behavior, rectal temperature and brain cAMP levels of rats: interaction with rolipram

The effects of systemically administered forskolin, a direct activator of the catalytic subunit of adenylate cyclase, on locomotor activity, rectal temperature and the incidence of grooming and head twitches were studied in rats. Forskolin, 0.1–25 mg/kg, decreased locomotor activity and lowered rectal temperature. The incidence of grooming and head twitches increased dose-dependently after forskolin, 6.25–25 mg/kg. The combination of a threshold dose of forskolin with a threshold dose of the cAMP-selective phosphodiesterase (PDE) inhibitor rolipram resulted in a marked, statistically significant enhancement of the behavioral, hypothermic and brain cAMP elevating effect. The present findings support the assumption that enhanced brain cAMP availability is associated with a characteristic behavioral syndrome in rats as was previously shown with dibutyryl cAMP or neurotropic cAMP-selective PDE inhibitors like rolipram, Ro 20-1724 or ICI 63 147.

Agents that elevate platelet cAMP stimulate the formation of phosphatidylinositol 4-phosphate in intact human platelets

The present study investigates the effect of compounds that are known to elevate cAMP on the phospholipid metabolism of platelets. Prostaglandin E 1, forskolin and isobutylmethylxanthine induce an increase in [ 32P]-phosphatidylinositol 4-phosphate (PIP) in platelets prelabelled with [ 32P]orthophosphate. Possible roles of this phenomenon are discussed in view of the inhibitory effect of cAMP elevation on platelet activation.

Activation of macrophage adenylate cyclase by stimulants of the oxidative burst and by arachidonic acid—Two distinct mechanisms

The effect of agents stimulating the oxidative burst (OB) in oil-elicited guinea pig peritoneal macrophages (MPs) on cyclic adenosine 3′,5′-monophosphate (cAMP) levels was examined. We found that: (i) Phorbol myristate acetate (PMA), the Ca 2+ ionophore A23187, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) and opsonized zymosan, elevated cAMP levels two- to fivefold; (ii) the biologically inactive PMA analog, 4-O-methyl-PMA, was proportionally less effective than PMA in stimulating cAMP accumulation; (iii) increased levels of cAMP were evident after 10 min of incubation with the stimulants, in the presence of the phosphodiesterase inhibitor 3-isobutyl methylxanthine (IBX); (iv) basal cAMP levels in MPs increased proportionally with the extracellular Ca 2+ concentration; (v) the cAMP-elevating effect of all stimulants (with the exception of A23187) was more pronounced in low Ca 2+ media, associated with lower basal cAMP levels. A23187 did not elevate cAMP levels in the absence of extracellular Ca 2+; (vi) short-term incubation of MPs with arachidonic acid and with the arachidonic acid precursor, linoleic acid, induced an increase in the level of cAMP; (vii) the elevations in cAMP levels induced by OB stimulants were enhanced, not blocked, by mepacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin or aspirin, demonstrating that prostaglandin (PG) synthesis was not involved; (viii) the cAMP-elevating effect of arachidonic and linoleic acids was blocked by ETYA and indomethacin, indicating that it was mediated by PGs. The mechanism by which OB stimulants elevate cAMP levels could not be determined but changes in the cellular level of Ca 2+ seem to play a pivotal role.

The inhibitory effect of paraquat on histamine and isoproterenol induced changes of cyclic nucleotides in rat lung slices

The incubation of rat lung slices with paraquat ion (10(-4) M) had no effect on cAMP and cGMP levels of the rat lung slices. The preincubation with the same concentration of paraquat inhibited the cAMP elevating effect of histamine (10(-5) M) and isoproterenol (10(-5) M) and reduced the cGMP level to approximately 50% of the level obtained without preincubation with paraquat.

Influence of papaverine and isoprenaline on contractility and cyclic AMP level of left guinea-pig atria at different rates of beat

Responses to papaverine and isoprenaline on cyclic AMP and contractility were investigated in left atria of guinea-pigs driven at a rate of 0.5 or 2 Hz. The dissociation of the effects of papaverine on contractility and cAMP seems to be related to a calcium antagonistic property which—in addition to the inhibition of phosphodiesterase—comes into play at higher rates of beat and inhibits the effects of elevated cAMP on myocardial contractility.