cAMP Modulation Research Articles

Bovine oocytes as a model system for the study of Protein Kinase C (PKC) signaling during mammalian oocyte in vitro maturation (IVM)

Bovine oocytes as a model system for the study of Protein Kinase C (PKC) signaling during mammalian oocyte in vitro maturation (IVM)

Circadian Regulation of cGMP-Gated Channels of Vertebrate Cone Photoreceptors: Role of cAMP and Ras

Circadian oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Raf also blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP --> PKA --> Ras --> B-Raf --> MEK --> Erk --> --> CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires >1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting.

S4 movement in a mammalian HCN channel.

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning.

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.

Adenylyl cyclase expression and modulation of cAMP in rat taste cells.

cAMP is a second messenger implicated in sensory transduction for taste. The identity of adenylyl cyclase (AC) in taste cells has not been explored. We have employed RT-PCR to identify the AC isoforms present in taste cells and found that AC 4, 6, and 8 are expressed as mRNAs in taste tissue. These proteins are also expressed in a subset of taste cells as revealed by immunohistochemistry. Alterations of cAMP concentrations are associated with transduction of taste stimuli of several classes. The involvement of particular ACs in this modulation has not been investigated. We demonstrate that glutamate, which is a potent stimulus eliciting a taste quality termed umami, causes a decrease in cAMP in forskolin-treated taste cells. The potentiation of this response by inosine monophosphate, the lack of response to d-glutamate, and the lack of response to umami stimuli in nonsensory lingual epithelium all suggest that the cAMP modulation represents umami taste transduction. Because cAMP downregulation via ACs can be mediated through Galpha(i) proteins, we examined the colocalization of the detected ACs with Galpha(i) proteins and found that 66% of AC8 immunopositive taste cells are also positive for gustducin, a taste-specific Galpha(i) protein. Whether AC8 is directly involved in signal transduction of umami taste remains to be established.

Epithelial sodium channels expressed in Xenopus oocytes are activated by cyclic-AMP

Background. The regulation of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis. The effect of cyclic AMP (cAMP) on ENaC activity is tissue-specific, and is controversial when ENaC is ex-pressed in Xenopus oocytes. Methods. The modulation of ENaC by cAMP in oocytes expressing human or rat ENaC was performed with two-electrode voltage clamping. Results. 250 μM 8-(4-chlorophenylthio)-adenosine 3′, 5′-cyclic monophosphate (8-CPT-cAMP) added to the bath significantly increased normalized amiloride-sensitive currents within 60 s in oocytes expressing human α, β, and γ subunits (5 ng cRNA each). The cAMP effect was dose-dependent and was partially inhibited by 200 μM Rp-CPT-cAMP, a competitive cAMP antagonist. A transient effect of 8-CPT-cAMP on rat ENaC activity was also observed. Oocytes expressing rat α subunits with γ subunits (which have a putative protein kinase A phosphorylation site) showed similar increases in amiloride-sensitive current with 250 μM 8-CPT-cAMP, while oocytes expressing rat α subunits with β subunits were not activated by 8-CPT-cAMP. Further, rat ENaC (but not human ENaC)-expressing oocytes were not activated by cAMP when oocytes were continuously superfused during electrophysiological recordings, suggesting that rat ENaC activation by cAMP is dependent upon the condition of oocytes during cAMP stimulation. Conclusion. The present results suggest that ENaC expressed in Xenopus oocytes can be activated by cAMP, and that the γ subunit confers sensitivity to cAMP modulation of rat ENaC activity.

Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes.

Recently, we established that cellular contractions increase long-chain fatty-acid (FA) uptake by cardiac myocytes. This increase is dependent on the transport function of an 88 kDa membrane FA transporter, FA translocase (FAT/CD36), and, in analogy to skeletal muscle, is likely to involve its translocation from an intracellular pool to the sarcolemma. In the present study, we investigated whether cAMP-dependent signalling is involved in this translocation process. Isoproterenol, dibutyryl-cAMP and the phosphodiesterase (PDE) inhibitor, amrinone, which markedly raised the intracellular cAMP level, did not affect cellular FA uptake, but influenced the fate of intracellular FAs by directing these to mitochondrial oxidation in electrostimulated cardiac myocytes. The PDE inhibitors 3-isobutyl-1-methylxanthine, milrinone and dipyridamole each significantly stimulated FA uptake as well as intracellular cAMP levels, but these effects were quantitatively unrelated. The stimulatory effects of these PDE inhibitors were antagonized by sulpho- N -succinimidylpalmitate, indicating the involvement of FAT/CD36, albeit that the different PDE inhibitors use different molecular mechanisms to stimulate FAT/CD36-mediated FA uptake. Notably, 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification, and dipyridamole induces translocation of FAT/CD36 to the sarcolemma. Elevation of intracellular cGMP, but not of cAMP, by the PDE inhibitor zaprinast did not have any effect on FA uptake and metabolism by cardiac myocytes. The stimulatory effects of PDE inhibitors on cardiac FA uptake should be considered when applying these agents in clinical medicine.

Changes in cAMP Affinity Modulate HCN Channel Activity

Wang et al. describe a mechanism whereby activity-dependent changes in adenosine 3′,5′-monophosphate (cAMP) binding affinity, rather than changes in cAMP concentration, can modulate activity of the hyperpolarization-activated, cyclic nucleotide-gated, cation nonselective (HCN) channel and can help regulate rhythmic firing. HCN channels are involved in pace-making and are modulated by cAMP, which facilitates channel opening. This has been modeled as an allosteric interaction in which channel opening is coupled with a transition to a conformation that enhances cAMP binding. Wang et al. used inside-out patch-clamp and voltage-clamp analyses of oocytes expressing HCN2 channel mRNA to test this model. Concentrations of cAMP ≥10 μM accelerated current activation and shifted the voltage dependence to more positive potentials, whereas 10 nM cAMP produced a slow phase of activation, consistent with preferential binding of cAMP to the open channel, shifting the equilibrium between closed and open unbound channels and eliciting a delayed phase of channel opening. Activation kinetics of channels in inside-out patches without cAMP fit a single exponential time course; a second exponential was apparent when cAMP was present. Activation kinetics of channels in intact oocytes resembled those of cell-free patches in low concentrations of cAMP; mutant channels that were unable to bind cAMP, however, showed only a single exponential component. Normal--but not mutant--channels showed "memory" and responded to a train of hyperpolarizations with a prolonged tail of channel opening that lasted for tens of seconds. Modeling studies indicated that cAMP modulation of neuronal HCN channels could play an important role in regulating rhythmic activity. J. Wang, S. Chan, M. F. Nolan, S. A. Siegelbaum, Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: Signaling through dynamic allosteric coupling. Neuron 36 , 451-461 (2002). [Online Journal]

Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells.

We have previously reported that the pigmented rabbit conjunctiva is a Cl- secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl- channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl- channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 microM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl- concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl- reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl- conductance in pigmented rabbit conjunctival epithelial cells.

The effects of cAMP modulation upon the adhesion and respiratory burst activity of immune complex-stimulated equine neutrophils

Toxic products such as reactive oxygen intermediates released by activated polymorphonuclear neutrophil (PMN) have an important role in the pathophysiology of diseases associated with the deposition of immune complexes (IC) in tissues. IC-induced activation of PMN requires adhesion mediated by integrin adhesion receptors. Of the integrins expressed on PMN, the β 2 family has been found to be of particular importance for activation of PMN by IC. β 2 Integrin ligand binding must be activated to enable adhesion to IC. Both activating and inhibitory signals regulate β 2 integrin ligand avidity and adhesion. The second messenger cyclic adenosine monophosphate (cAMP) has been demonstrated to inhibit the activation of PMN in response to a variety of stimuli. The purpose of this study is to test the hypothesis that cAMP-dependent signals inhibit β 2 integrin-dependent adhesion of equine PMN to immobilized IC and subsequent adhesion-dependent activation of respiratory burst activity. Treatment of equine PMN with β 2 adrenergic agonists isoproterenol or clenbuterol, which trigger an increase in intracellular cAMP concentration, inhibited adhesion of equine PMN to IC in a dose dependent manner. Similarly, inhibition of cAMP hydrolysis by the non-specific phosphodiesterase (PDE) inhibitor pentoxifylline and the PDE 4-specific inhibitor rolipram inhibited adhesion of equine PMN to IC. Elevation of intracellular cAMP levels with pentoxifylline, clenbuterol and rolipram also inhibited IC-induced activation of respiratory burst activity in equine PMN. Importantly, co-treatment of equine PMN with rolipram and either β 2 adrenergic agonist synergistically inhibited both the adhesion of equine PMN to IC as well as the subsequent respiratory burst activity.

Regulation of hyperpolarization-activated HCN channel gating and cAMP modulation due to interactions of COOH terminus and core transmembrane regions.

Members of the hyperpolarization-activated cation (HCN) channel family generate HCN currents (I(h)) that are directly regulated by cAMP and contribute to pacemaking activity in heart and brain. The four different HCN isoforms show distinct biophysical properties. In cell-free patches from Xenopus oocytes, the steady-state activation curve of HCN2 channels is 20 mV more hyperpolarized compared with HCN1. Whereas the binding of cAMP to a COOH-terminal cyclic nucleotide binding domain (CNBD) markedly shifts the activation curve of HCN2 by 17 mV to more positive potentials, the response of HCN1 is much less pronounced (4 mV shift). A previous deletion mutant study suggested that the CNBD inhibits hyperpolarization-gating in the absence of cAMP; the binding of cAMP shifts gating to more positive voltages by relieving this inhibition. The differences in basal gating and cAMP responsiveness between HCN1 and HCN2 were proposed to result from a greater inhibitory effect of the CNBD in HCN2 compared with HCN1. Here, we use a series of chimeras between HCN1 and HCN2, in which we exchange the NH(2) terminus, the transmembrane domain, or distinct domains of the COOH terminus, to investigate further the molecular bases for the modulatory action of cAMP and for the differences in the functional properties of the two channels. Differences in cAMP regulation between HCN1 and HCN2 are localized to sequence differences within the COOH terminus of the two channels. Surprisingly, exchange of the CNBDs between HCN1 and HCN2 has little effect on basal gating and has only a modest one on cAMP modulation. Rather, differences in cAMP modulation depend on the interaction between the CNBD and the C-linker, a conserved 80-amino acid region that connects the last (S6) transmembrane segment to the CNBD. Differences in basal gating depend on both the core transmembrane domain and the COOH terminus. These data, taken in the context of the previous data on deletion mutants, suggest that the inhibitory effect of the CNBD on basal gating depends on its interactions with both the C-linker and core transmembrane domain of the channel. The extent to which cAMP binding is able to relieve this inhibition is dependent on the interaction between the C-linker and the CNBD.

Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP and Ca2.

• Modulators of cAMP, calcium and G proteins were used to treat bean (Phaseolus vulgaris) cells before addition of an elicitor from Colletotrichum lindemuthianum in order to elucidate the early steps of signal transduction leading to the production of the apoplastic oxidative burst. • Hydrogen peroxide production by elicited bean cells was monitored with luminol-or xylenol-orange-based assays. • Pretreatment with forskolin, dibutyryl cAMP or the Ca2+ ionophore A23187 enhanced the production of reactive oxygen species (ROS). The Ca2+ channel blocker, verapamil, and the calmodulin antagonist W7 led to a decreased oxidative burst and cancelled the dibutyryl cAMP effect. The production of ROS was increased by cholera toxin (CTX), an activator of G proteins. • Thus, an increase of cytosolic calcium ([Ca2+ ]cyt ) mediated through an increased level of cAMP is required for ROS production. The data support a role for G proteins and cAMP in extracellular alkalinization and Ca2+ influx, possibly in the provision of a reductant, which with the extracellular peroxidase, are required for the apoplastic oxidative burst.

Molecular mechanism of cAMP modulation of HCN pacemaker channels.

Hyperpolarization-activated cation channels of the HCN gene family contribute to spontaneous rhythmic activity in both heart and brain. All four family members contain both a core transmembrane segment domain, homologous to the S1-S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel--important for visual and olfactory signalling and for cardiac repolarization, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential, thereby accelerating rhythmogenesis. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms.

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C)

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [ 125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200–300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.

Expression and Up-Regulation of Alternatively Spliced Transcripts of Melastatin, a Melanoma Metastasis-Related Gene, in Human Melanoma Cells

Loss of expression of a novel suppressor of metastasis, melastatin (MLSN1), has recently been reported to correlate with metastatic potential of melanoma cells. Using differential display analysis, we identified MLSN1 among genes overexpressed in pigmented metastatic human melanoma cells treated with the differentiation inducer hexamethylene bisacetamide (HMBA). In this study, we show that multiple short transcripts of MLSN1 are present in melanocytes and pigmented metastatic melanoma cell lines while the full-length 5.4-kb mRNA is detectable only in melanocytes. Treatment of pigmented melanoma cells with the differentiation-inducing agent, HMBA, results in up-regulation of the 5.4-kb MLSN1 mRNA as well as short RNAs. Analysis of a panel of nonpigmented primary and metastatic melanoma cell lines showed weak expression of a 1.8-kb mRNA in a few melanoma cell lines. Northern blot and RT-PCR analyses with DNA probes and oligonucleotide primers that correspond to distinct regions of full-length MLSN1 mRNA indicated that the short transcripts contained sequences corresponding primarily to either 5′- or 3′-end of the 5.4-kb mRNA. HMBA appears to up-regulate MLSN1 transcripts derived mainly from the 5′-end. Modulators of cAMP and protein kinase C pathways had no significant effect on MLSN1 expression. Our data show that multiple MLSN1 transcripts, both constitutively expressed and inducible, are present in cultured pigmented melanoma cells, and suggest that MLSN1 expression can be regulated at the level of both transcription and mRNA processing.

Enhancement of the cAMP-induced apolipoprotein-mediated cellular lipid release by calmodulin inhibitors W7 and W5 from RAW 264 mouse macrophage cell line cells.

Apolipoprotein (apo) A-I generates high-density lipoprotein (HDL) by removing cellular cholesterol and phospholipid on the interaction with cells as a main source of plasma HDL. The reaction is induced by dibutylyl cyclic (dbc) adenosine monophosphate (AMP) in RAW 264, mouse macrophage cell line cells, and we investigated its pharmacologic modulation using this cell model. Release of cellular cholesterol and choline phospholipid by apoA-I was increased 9.9 and 4.2 times, respectively, by pretreatment of the cells with 300 microM dbcAMP for 24 h. Calmodulin inhibitors, W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) and W5 (N(6-aminohexyl)-1-naphthalenesulfonamide), increased the apoA-I-mediated lipid release by 3 times from the dbcAMP-treated cells. The optimal drug concentrations (80 and 160 microM for W7 and W5, respectively) were not parallel with those reported for in vitro calmodulin inhibition (IC50, 28 and 240 microM for W7 and W5, respectively, toward phosphodiesterase activity), and in fact 40 microM W7 showed much stronger intracellular calmodulin inhibition than did 300 microM W5 using S7AAS2, a fluorescent peptide probe. Other calmodulin inhibitors such as amitriptyline, chlorpromazine, and trifluoperazine showed no effect on the apoA-I-mediated cholesterol release. In contrast to these results, neither dbcAMP nor W7 influenced the diffusion-mediated nonspecific cholesterol efflux to lipid microemulsion. We concluded that W7 and W5 increased the interaction of apoA-I with RAW 264 cells to generate more HDL. The effect did not seem directly correlated to their cal modulin inhibition or modulation of cAMP and protein kinase C.

Modulation of cAMP and phosphodiesterase activity by flavonoids which induce spore germination of Nectria haematococca MP VI (Fusarium solani)

Flavonoids exuded from legume roots stimulate spore germination of a number of soilborne fungi which interact with these plants, and so may act as host cues to initiate interaction. Macroconidia of Nectria haematococca MPVI (anamorph Fusarium solani), a pathogen of pea (Pisum sativum), germinate in response to the same flavonoids which induce nod gene expression in pea-specific rhizobia. Pisatin, the isoflavonoid phytoalexin of pea, also induces germination. The present study found that cAMP levels in macroconidia were transiently induced by flavonoid treatment. Combined with previous pharmacological studies, this indicates that flavonoid signalling utilizes the cAMP pathway. A hypothesis that flavonoids modulate cAMP levels through direct inhibition of N. haematococca cAMP phosphodiesterase was tested. A low KMcAMP phosphodiesterase activity from macroconidia was tested for inhibition by 10 flavonoids. Naringenin, a strong inducer of germination, was a strong inhibitor of phosphodiesterase (apparent Ki33 μm). There was a general correlation between strength of induction and inhibition of phosphodiesterase. Pisatin, structurally distinct from the others, appeared to be an exception to this trend. The results suggest that the ability of specific flavones and flavanones to inhibit cAMP phosphodiesterase is a potential mechanism through which they can induce cAMP levels and so promote germination.

The Biphasic Induction of p21 and p27 in Breast Cancer Cells by Modulators of cAMP Is Posttranscriptionally Regulated and Independent of the PKA Pathway

Cyclic AMP (cAMP) elevation affects growth arrest and differentiation in a wide variety of breast cell lines; however, the mechanisms associated with this process are poorly understood. Previous studies linked cAMP-mediated growth arrest in breast tumor cells to increased levels of cyclin kinase inhibitor (CKI), p21. In the present study we examined the role of cAMP-dependent protein kinase (PKA) on p21 and p27 induction in the breast cancer cell line, MDA-MB-157. The induction of the CKIs by modulators of cAMP such as cholera toxin (CT) + 1-isobutyl-3-methylxanthine (IBMX) and lovastatin fluctuates with biphasic kinetics (although the kinetics of CKI induction with CT + IBMX treatment are different from that of lovastatin) and is depicted by the periodic accumulation of lower molecular weight forms of p21 and p27 which also correlate with fluctuations in CDK2 activity. Using three different approaches we show that the cAMP-mediated induction of CKIs is independent of PKA activity. In the first approach we treated MDA-MB-157 cells with a variety of cAMP modulators such as CT + IBMX, and forskolin in the presence or absence of H-89, a potent PKA inhibitor. This analysis revealed that the cAMP activators were capable of inducing p21 even though PKA activity was completely eliminated. In the second approach PKA dominant negative stable clones of MDA-MB-157 treated with CT + IBMX or forskolin also resulted in p21 induction, in the absence of any PKA activity. Last, treatment of MDA-MB-157 cells with lovastatin, another known cAMP modulator which also causes growth arrest, resulted in the induction of p21 and p27 without any increase in PKA activity. Collectively, the above results suggest that the induction of p21 by cAMP is through a novel pathway, independent of PKA activity.

INDOMETHACIN DOES NOT INFLUENCE ALVEOLAR LIQUID CLEARANCE IN ANESTHETIZED SHEEP OR RATS

Active transport of sodium has been shown to be the predominant mechanism involved in alveolar liquid clearance. One regulatory mechanism involved in the modulation of this transport system is cAMP. Although it was initially thought that cAMP could directly modulate transepithelial Na+ transport, recent data suggest that this cAMP modulation could be secondary to the production of arachnidonic acid metabolites. The purpose of this study was thus to evaluate if prostaglandin products could have an indirect or direct role to play in lung liquid clearance. Addition of 10 5 M salmeterol, known to increase intracellular cAMP, to the instilled fluid in rats stimulated lung liquid clearance. However, addition of indomethacin did not influence the stimulating effect of salmeterol. Furthermore, addition of prostaglandin E2 to the instilled fluid did not stimulate alveolar fluid clearance. In order to determine if this response could be species related, we evaluated if indomethacin could modulate alveolar liquid clearance in sheep. Presence of cAMP and aminophylline stimulated lung liquid clearance in sheep, but indomethacin did not inhibit this response. The present study demonstrates that cyclooxygenase products are not involved in the modulation of basal or stimulated alveolar or lung liquid clearance in sheep or rats.