Role Of cAMP In Regulation Research Articles

CAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1 to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1 proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/- and p21Cip1-/- 3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase-specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase-specific antitumor agents.

Dominant role of cAMP in regulation of microvessel permeability.

We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (L(p)), and that the activation of cGMP-dependent pathways was a necessary step to increase L(p) in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal L(p) in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in L(p) due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 microM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in L(p) due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in L(p). These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner.

The intent of this work was to evaluate the role of cAMP in regulation of ciliary activity in frog mucociliary epithelium and to examine the possibility of cross talk between the cAMP- and Ca2+-dependent pathways in that regulation. Forskolin and dibutyryl cAMP induced strong transient intracellular Ca2+ concentration ([Ca2+]i) elevation and strong ciliary beat frequency enhancement with prolonged stabilization at an elevated plateau. The response was not affected by reduction of extracellular Ca2+ concentration. The elevation in [Ca2+]i was canceled by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Under those experimental conditions, forskolin raised the beat frequency to a moderately elevated plateau, whereas the initial strong rise in frequency was completely abolished. All effects were canceled by H-89, a selective protein kinase A (PKA) inhibitor. The results suggest a dual role for PKA in ciliary regulation. PKA releases Ca2+ from intracellular stores, strongly activating ciliary beating, and, concurrently, produces moderate prolonged enhancement of the beat frequency by a Ca2+-independent mechanism.

Gap Junctional Intercellular Communication of Bovine Luteal Cells from Several Stages of the Estrous Cycle: Effects of Cyclic Adenosine 3′,5′-Monophosphate1

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p < 0.01) but Rp-cAMPS (p < 0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p < 0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p < 0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p < 0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p < 0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p < 0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p < 0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p < 0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p < 0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p < 0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Cyclic AMP-phosphodiesterase IIIA1 inhibitors decrease cytosolic Ca 2+ concentration and increase the Ca 2+ content of intracellular storage sites in human platelets

The effect of cyclic AMP-phosphodiesterase (cAMP-PDE) inhibitors on Ca 2+ homeostasis in human platelets was studied using both quin-2 (2-(-bis-(acetylamino)-5-methyl-phenoxy)methyl-6-methoxy-8-bis-(acetylamino)quinoline) and chlorotetracycline (CTC) to measure changes in cytosolic Ca 2+ as well as changes in the amount of Ca 2+ accumulated in intracellular storage sites. At therapeutic concentrations (1 μM) milrinone and R 80 122 but not enoximone decreased the cytosolic Ca 2+ concentration in the resting platelet while the Ca 2+ content in intracellular stores was increased. These observations are in accord with the proposed mechanism of action of cAMP-PDE inhibitors on cardiomyocites and highlight the particular role of cAMP in regulation of Ca 2+ homeostasis.

Effect of cyclic AMP on the isolated human oocyte--cumulus complex.

Cyclic AMP (cAMP) is one of the key regulators of the resumption of oocyte meiosis (oocyte maturation) in several mammalian species. In this study we have examined the role for cAMP in regulation of spontaneous maturation of human oocytes. Oocytes were obtained in connection with gynaecological operations and cultured for 24 or 48 h. A significantly larger proportion (77%) of oocytes cultured in the presence of 1 mM dibuturyl-cAMP (dbcAMP) for 24 h were arrested in meiosis compared to oocytes cultured in control medium (25%). In addition, dbcAMP stimulated progesterone accumulation during 48 h of incubation and inhibited monolayer formation of the cumulus cells attached to the oocyte. The data presented in this study demonstrate that dbcAMP delayed the spontaneous maturation of human oocytes and stimulated progesterone production by the cumulus cells. These results indicate that cAMP may be important also for regulation of meiosis in human oocytes and confirm results obtained in other species.

Role of cAMP in regulation of activity of acid hydrolases of rat heart and liver during ischemia and after recirculation.

The interrelation between intracellular cAMP content and activity of lysosomal hydrolases was studied in rat liver and heart during ischemia of varying genesis and after recirculation. The activity of acid phosphatase (AP) and cathepsin D (CD) was determined in the fraction enriched with lysosomes (FEL) and in the supernatant fraction (SF) at 30,000 x g. Ischemia of isolated perfused heart of 20 to 60 min as described by Langendorff was accompanied by an increase in the SF/FEL ratio. Postischemic reperfusion resulted in a further increase in this ratio. In a terminal state induced by cardiac arrest of 10 min and within the first postresuscitation hours the SF/FEL ratio in the rat liver also increased. Processing of the liver FEL with 0.025% concentration of detergent Triton X-100 was also indicative of lability of lysosomal membranes during recirculation. The intracellular cAMP content changed differently. During ischemia of the myocardium, the cAMP level rose by 40 min and remained increased after 20 and 40 min of reperfusion. The cAMP content in the liver decreased after 10 min of circulatory arrest and increased in the postresuscitation period achieving its peak 4 h after resuscitation. Intra-abdominal injection of lyposomes with incapsulated cAMP to rats in the postresuscitation period and the study of the effect of dibutyryl-cAMP, caffeine and isoproterenol on the activity of acid hydrolases of ischemic heart and after postischemic reperfusion showed that an increase in the cAMP content achieved in various ways was conducive to stabilization of lysosomal membranes.

CAMP activates Cl −/HCO 3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK 1 cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na +-independent Cl −/HCO 3 − exchange, and by removal of extracellular Cl −. In addition, Bt 2 cAMP causes Cl − entry into LLC-PK 1 cells. These results suggest that cAMP activates Cl − transport, namely Na +-independent Cl −/HCO 3 − exchange, which participates in pH i regulation.

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical1stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an afterdischarge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.

Dissociation between the inhibitory and the stimulatory action of cAMP on maturation of rat oocytes

The possible mediatory role of cAMP in the induction of oocyte maturation by luteinizing hormone (LH) is not yet clear since evidence for both inhibitory and stimulatory actions of the nucleotide on the oocyte has been provided. To elucidate the role of cAMP in regulation of oocyte meiosis we tried in the present study to dissociate between the inhibitory and stimulatory action of this nucleotide on oocyte maturation. To induce maturation, oocytes enclosed by their follicles were transiently exposed to either dibutyryl cAMP (dbcAMP) or to the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Inhibition of maturation was obtained by the addition of the above agents to either follicle-enclosed oocytes incubated in the presence of LH or isolated cumulus-free oocytes that mature spontaneously in vitro. We found that inhibition of oocyte maturation is obtained by a relatively low dose of either dbcAMP or MIX while higher concentrations of these agents are required to induce oocyte maturation. Coupling of the oocyte to the cumulus cells, as expressed by the fraction of labeled uridine transferred from the cumulus cells to the oocyte following exposure of the follicle-enclosed cumulus-oocyte complex to MIX, was also determined. We found that uncoupling of the oocyte from the cumulus cells corresponded with the induction, but not inhibition of oocyte maturation, both by its concentration dependence and time-course. We suggest that cAMP has a dual role in regulation of oocyte maturation. Lower levels of the nucleotide act to maintain meiotic arrest, while elevated levels of cAMP mediate LH action to induce meiosis resumption.

Role of cAMP in regulation of hippocampal unit activityin vitro

Role of cAMP in regulation of hippocampal unit activityin vitro