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1.
In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca 2+ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca 2+] in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca 2+] in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca 2+] in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions. 相似文献
2.
Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low K m cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH 4C 1) cells in culture (ED 50=5·10 –8 M). This effect is due to a selective inhibition of the low K m cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca 2+/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the
i family ( G
i2 or G
k). Rolipram did not affect phosphoinositide metabolism (i.e. IP 3 accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.Long term incubation of GH 4C 1 cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of -receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in -adrenoceptor AC downregulation. These features are not shared by the other drugs tested. 相似文献
3.
An adenylate cyclase activity of 16.02±1.03 pmol cAMP produced min −1 (mg protein) −1 was detected in a cell homogenate of Dunaliella viridis, a unicellular halotolerant green alga. It was present in both the membrane fraction and soluble fraction separated from
the homogenate. Adenylate cyclase activity in the homogenate was activated by 1μM GTPγS but not by Ca 2++calmodulin, suggesting this enzyme to be regulated by a G-protein. A phosphodiesterase activity of 23.12±15.03 pmol cAMP
decomposed min −1 (mg protein) −1 was found in the homogenate. These activities suggest the presence of a cAMP mediated signal transduction system in Dunaliella. Cells, transferred from 1.7 M NaCl medium to 1 M NaCl, showed rapid increase in cAMP within 2 min to about 1.5 times the
original concentration (from 2.4±0.2 to 3.9±0.2 pmol per 10 8 cells) which was recovered in 30 min. 相似文献
4.
BackgroundThere is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (I Ca) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β 1-adrenoceptor (β 1-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on I Ca and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes. MethodsAdult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). I Ca was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β 1-AR were stimulated with the physiological agonist norepinephrine (100 μM). ResultsIn adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% ( n = 10/9, mean ± SEM, number of cells/number patients) and I Ca by +10.4 ± 1.5 pA/pF ( n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and I Ca (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and I Ca + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and I Ca + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on I Ca. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and I Ca, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and I Ca + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on I Ca (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET. ConclusionOur results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller I Ca responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes. 相似文献
5.
The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic- S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A ( KI = 43.4 ± 7.2 μM and kcart = 0.007 ± 0.0006 min −1). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS ( Kd = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD +, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes. 相似文献
6.
Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M 2AChR pharmacological profile. Therefore, a novel Ca 2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58?kDa) being the 58?kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [ 3H]cGMP and [ 3H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg 2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC 50 of 4.9 and 4.6?μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M 2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed. 相似文献
7.
The mode of the developmental expression of adenylate cyclase (AC) and phosphodiesterase (PDE) in D melanogaster indicates that PDE plays the major role in the maintenance of a certain level of cAMP in postembryonic development, while both enzymes function in concert in imago. The ts-mutants ts155 and ts622, characterized upon their isolation as having an increased cAMP content and normal PDE activity, manifest high levels of AC activity from the third day of imago life. The levels of PDE activity characteristic for adult mutants with altered enzyme activity (low in ts66 and ts980, high in ts398) are manifested in ts980 from larval instar II, and from the larval instar III in ts398 and ts66. Data on the dependence of PDE activity in adults upon temperature of incubation, being in agreement with the expectations for a ts-mutation in a gene coding for a form of PDE in case of ts66, suggest that ts398 affects not the enzyme-coding gene but rather one for an activator protein. The fact that in ts398 (the polyphasic ts-lethal mapping to 1-38.9) 1) AC activity is somewhat higher than normal at 22°C and is readily activated at 29°C, 2) activity of PDE-I assayed in heat-pretreated homogenates is higher than normal, 3) that boiled extracts of ts398 are potent activators of the wild type and of its own PDE-I indicates that it is a mutation affecting calmodulin, which is known to be stable at boiling and capable of activating both AC and PDE-I. Data on Ca 2+ and EGTA effects suggest that the mutation presumably increases Ca 2+-binding activity of calmodulin, ts980 and ts622, in which ts-lethality could be produced only by certain doses of haloperidol and triftazine, appear to be lethal in compounds with ts398, thus indicating that these mutations could affect the same calmodulin-controlling gene. 相似文献
8.
Rat forebrain- and heart-derived mRNA were used to express Ca 2+ channels in Xenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca 2+ channel currents ( I
Ba, 40 mM Ba 2+ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which
allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directed I
Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by
70% by 1 μM of ω-conotoxin GVIA, while I
Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directed I
Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator
forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac” I
Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM),
as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular
perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted
no effect on the basal level of I
Ba, suggesting that the expressed Ca 2+ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and
heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration
(2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but
not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA into Xenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca 2+ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific,
since N-type Ca 2+ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to
cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of
functional significance of phosphorylation. 相似文献
9.
The Ca 2+ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca 2+ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca 2+ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca 2+ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca 2+ and cAMP signaling and the function of IRBIT (IP 3 receptors binding protein release with IP 3) as a third messenger that mediates the synergistic action of the Ca 2+ and cAMP signaling. 相似文献
11.
Background Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca 2+ concentration ([Ca 2+] i) that is composed of liberation of stored Ca 2+ and extracellular Ca 2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca 2+] i. 相似文献
12.
The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D, and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca 2+ and calmodulin. Nevertheless, the Ca 2+—calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K m values were found for PDE from groups C, D, and I, whereas the V max2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats. © 1996 Wiley-Liss, Inc. 相似文献
13.
Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca 2+] o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca 2+] o enhances adipocyte accumulation.We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca 2+ and Sr 2+) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca 2+] o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1 μM forskolin and 200 nM IBMX) and a cAMP analog (10 μM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca 2+] o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca 2+] o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25–40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca 2+] o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP. 相似文献
14.
Studies of bovine hypothalamic cyclic nucleotide phosphodiesterase (PDE) indicate the presence of several peaks of PDE activity,
distinguishable by DEAE-cellulose column chromatography, displaying different substrate specificities, kinetic behavior, and
regulatory properties. Evidence is presented that chromatographically separated forms of PDE activity are subject to control
by Ca 2+-calmodulin, cyclic nucleotides, limited proteolysis, reagents affecting sulfhydryl groups, and neurohormone “C”—one of several
new cardioactive compounds isolated from hypothalamic magnocellular nuclei of animals—in a complex substrate-specific and
concentration-dependent manner. Of particular interest is the finding that each of the forms of cGMP PDE, being Ca 2+/calmodulin-dependent, possesses sensitivity to activation by cAMP, especially under conditions favoring the oxidation of
thiol groups of PDE, resulting in a loss in responsiveness of the enzyme to the activation by calmodulin. This effect appears
to be relatively stable but readily reversible by sulfhydryl reducing reagents, which restore both the cGMP PDE sensitivity
to competitive inhibition by cAMP and the responsiveness of the enzyme to activation by calmodulin. A reinterpretation of
the regulatory properties of multiple forms of PDE is proposed.
Special Issue dedicated to Dr. Eugene Kreps. 相似文献
15.
Oridonin is a diterpenoid isolated from medicinal herb Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), which has an allelopathic effect on plants. Phospholipase C (PLC1) and hydrogen peroxide (H2O2) are involved in many biotic or abiotic stress responses. Using the 16-day-old seedlings of Arabidopsis thaliana ecotype (WT) and PLC1-deficient mutant (plc1) as materials (treated with 10 μM or 60 μM oridonin for 72 h), the effect of oridonin on root growth regulating by PLC1 and H2O2 was investigated. The results showed that the promoting of root growth was about 6.9% at 10 μmol L?1 oridonin and the inhibiting of root growth was about 19.73% at 60 μmol L?1 oridonin in WT, the inhibiting of root growth was about 10.5% and 41.2% at 10 mol L?1 and 60 mol L?1 oridonin, respectively, in plc1. The expression of ARR1, ARR12, and AHK3 was promoted at low concentrations of oridonin and inhibited at high concentrations in WT, whereas the expression of ARR1 and ARR12 was inhibited with the increase of oridonin concentration in plc1. This suggested that PLC1 was involved in the root growth regulation of oridonin. H2O2 was promoted by oridonin with concentration dependence pattern in root cells. Oridonin increased the activity of antioxidant enzymes in both WT and plc1, but the activity of antioxidant enzymes in plc1 was lower than WT. This indicated that PLC1 involved in the activation of antioxidant enzymes promoted by the oridonin. Exogenous CaCl2 facilitated the accumulation of H2O2 in both WT and plc1. And the H2O2 of WT was obviously higher than that of plc1. The root growth of WT was inhibited by CaCl2 with the increase of oridonin. However, there is no effect of CaCl2 on the root growth in plc1. This reflected that PLC1 positively involved in the regulation of Ca2+ on the H2O2 and the inhibition effect of Ca2+ on the root growth under oridonin treatment. PA promoted the H2O2 and suppressed the root growth under oridonin treatment in both WT and plc1. In plc1, PA facilitated the root growth with no oridonin and inhibited the root growth with the increase of oridonin. This reflected that PLC1 positively regulated the promotion effect of PA on the root growth under high oridonin treatment. PLC1 mediated oridonin (10 and 60 mol L?1) to regulate H2O2 levels in A. thaliana seedlings, thereby regulating root tip cell morphology and mitosis. These results demonstrated that PLC1 mediated the low-promotion and high-inhibition effect of oridonin on the root growth in A. thaliana by regulating the concentrations of Ca2+ and PA, and further affecting the intracellular H2O2 level. 相似文献
16.
The elevation of [cAMP] i is an important mechanism of
platelet inhibition and is regulated by the opposing activity of adenylyl
cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a
variety of platelet agonists, including thrombin, significantly enhance the
activity of PDE3A in a phosphorylation-dependent manner. Stimulation of
platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient
phosphorylation of PDE3A on Ser 312, Ser 428,
Ser 438, Ser 465, and Ser 492, in parallel with
the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation
and activation of PDE3A required the activation of PKC, but not of PI3K/PKB,
mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in
phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent
manner. This was further supported by the finding that IGF-1, which strongly
activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation
also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In
contrast, cAMP-elevating agents such as PGE 1 and forskolin-induced
phosphorylation of Ser 312 and increased PDE3A activity, but did not
stimulate 14-3-3 binding. Finally, complete antagonism of
PGE 1-evoked cAMP accumulation by thrombin required both
G i and PKC activation. Together, these results demonstrate that
platelet activation stimulates PKC-dependent phosphorylation of PDE3A on
Ser 312, Ser 428, Ser 438, Ser 465,
and Ser 492 leading to a subsequent increase in cAMP hydrolysis and
14-3-3 binding.Upon vascular injury, platelets adhere to the newly exposed subintimal
collagen and undergo activation leading to platelet spreading to cover the
damaged region and release of thrombogenic factors such as ADP and thromboxane
A 2. In addition, platelets are activated by thrombin, which is
generated as a result of activation of the coagulation pathway, and stimulates
platelets by cleaving the protease-activated receptors
(PAR), 2
PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen
binding sites on integrin α IIbβ 3 resulting in
platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C
β (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a
subsequent increase in [Ca 2+] i and activation
of protein kinase C (PKC). Protein kinase C contributes to platelet activation
both directly, through affinity regulation of the fibrinogen receptor,
integrin α IIbβ 3
( 1), and indirectly by
enhancing degranulation ( 2).
Thrombin also stimulates activation of PI 3-kinases and subsequent generation
of PI ( 3,
4,
5) trisphosphate and PI
( 3,
4) bisphosphate
( 3), which recruit protein
kinase B (PKB) to the plasma membrane where it becomes phosphorylated and
activated.Platelet activation is opposed by agents that raise intracellular
3′-5′-cyclic adenosine monophosphate
([cAMP] i). cAMP is a powerful inhibitory second messenger
that down-regulates platelet function by interfering with Ca 2+
homeostasis, degranulation and integrin activation
( 4). Synthesis of cAMP is
stimulated by mediators such as prostaglandin I 2 (PGI 2),
which bind to G s-coupled receptors leading to activation of
adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which
inhibits the elevation of cAMP indirectly via autocrine activation of the
G i-coupled ADP receptor P2Y 12. cAMP signaling is
terminated by hydrolysis to biologically inert 5′-AMP by
3′-phosphodiesterases. Platelets express two cAMP phosphodiesterase
isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most
abundant isoform in platelets and has a ∼250-fold lower
Km for cAMP than PDE2
( 4). As a consequence of these
properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly
at resting levels. The importance of PDE3A in platelet function is further
emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone
raise basal cAMP levels and strongly inhibit thrombin-induced platelet
activation ( 5). Furthermore,
PDE3A -/- mice demonstrate increased resting levels of platelet cAMP
and are protected against a model of pulmonary thrombosis
( 6). In contrast, the PDE2
inhibitor EHNA has no significant effect on cAMP levels and platelet
aggregation ( 7,
8). The activity of PDE3A is
therefore essential to maintain low equilibrium levels of cAMP and determine
the threshold for platelet activation
( 7).Like its paralogue PDE3B, it has recently become clear that PDE3A activity
can be positively regulated by phosphorylation in platelets and human oocytes
( 9,
10). There is some evidence
that PKB may be involved in this regulation, although the phosphorylation
sites are poorly characterized. In contrast, phosphorylation of PDE3A in HeLa
cells was stimulated by phorbol esters and blocked by inhibitors of PKC
( 11). In this study, we aimed
to identify the signaling pathways and phosphorylation sites that are involved
in regulation of platelet PDE3A. Here, we show strong evidence that PKC, and
not PKB, is involved in agonist-stimulated PDE3A phosphorylation on
Ser 312, Ser 428, Ser 438, Ser 465,
and Ser 492, leading to an increase in PDE3A activity, 14-3-3
binding and modulation of intracellular cAMP levels. 相似文献
17.
In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic β-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic β-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca 2+) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca 2+ oscillations in the β-cell. Slow Ca 2+ oscillations (<1 min –1) produce low-frequency cAMP oscillations, and faster Ca 2+ oscillations (>3–4 min –1) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca 2+ oscillations. In contrast, observed antiphasic Ca 2+ and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca 2+-dependent AC and PDE activation in coupling of Ca 2+ and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 β-cells. adenylyl cyclase; calcium ion; glucagon-like peptide 1; modeling; oscillations 相似文献
18.
Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca 2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca 2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca 2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca 2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca 2 + stimulation of oxidative phosphorylation. 相似文献
19.
Abstract MC exhibits A 1 and A 2 receptors with opposite actions on cAMP formation and 45Ca 2+ uptake. ADO 10 ?4 M activated both second messengers, but neither A 1 nor A 2 receptors seem to be involved in these ADO-induced effects. 相似文献
20.
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby participating in regulation
of the intracellular concentrations of these second messengers. The PDE1 family is defined by regulation of activity by calcium
and calmodulin. We have cloned and characterized the mouse PDE1B gene, which encodes the 63-kDa calcium/calmodulin-dependent PDE (CaM-PDE), an isozyme that is expressed in the CNS in the
olfactory tract, dentate gyrus, and striatum and may participate in learning, memory, and regulation of phosphorylation of
DARPP-32 in dopaminergic neurons. We screened an I-129/SvJ mouse genomic library and identified exons 2–13 of the PDE1B gene that span 8.4 kb of genomic DNA. Exons range from 67 to 205 nucleotides and introns from 91 to 2250 nucleotides in length.
Exon 1 was not present in the 3 kb of genomic DNA 5′ to exon 2 in our clones. The mouse PDE1B gene shares many similar or identical exon boundaries as well as considerable sequence identity with the rat PDE4B and PDE4D genes and the Drosophila dunce cAMP-specific PDE gene dnc, suggesting that these genes all arose from a common ancestor. Using fluorescence in situ hybridization, we localized the
PDE1B gene to the distal tip of mouse Chromosome (Chr) 15.
Received: 10 November 1997 / Accepted: 12 March 1998 相似文献
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