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cAMP Analog (camp + analog)
Selected AbstractsModulation of perch connexin35 hemi-channels by cyclic AMP requires a protein kinase A phosphorylation siteJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Georgia Mitropoulou Abstract Retinal neurons are coupled via gap junctions, which function as electrical synapses that are gated by ambient light conditions. Gap junctions connecting either horizontal cells or AII amacrine cells are inhibited by the neurotransmitter dopamine, via the activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway. Fish connexin35 (Cx35) and its mouse ortholog, Cx36, are good candidates to undergo dopaminergic modulation, because they have been detected in the inner plexiform layer of the retina, where Type II amacrine cells establish synaptic contacts. We have taken advantage of the ability of certain connexins to form functional connexons (hemi-channels), when expressed in Xenopus oocytes, to investigate whether pharmacological elevation of cAMP modulates voltage-activated hemi-channel currents in single oocytes. Injection of perch Cx35 RNA into Xenopus oocytes induced outward voltage-dependent currents that were recorded at positive membrane potentials. Incubation of oocytes with 8-bromoadenosine 3,,5,-cyclic monophosphate (8-Br-cAMP), a membrane permeable cAMP analog, resulted in a dose-dependent and reversible inhibition of hemi-channel currents at the more positive voltage steps. In contrast, treatment with 8-Br-cAMP did not have any effect on hemi-channel currents induced by skate Cx35. Amino acid sequence comparison of the two fish connexins revealed, in the middle cytoplasmic loop of perch Cx35, the presence of a PKA consensus sequence that was absent in the skate connexin. The results obtained with two constructs in which the putative PKA phosphorylation site was either suppressed (perch Cx35R108Q) or introduced (skate Cx35Q108R) indicate that it is responsible for the inhibition of hemi-channel currents. These studies demonstrate that perch Cx35 is a target of the cAMP/PKA signaling pathway and identify a consensus PKA phosphorylation site that is required for channel gating. © 2003 Wiley-Liss, Inc. [source] Role of calcium in the gating of isoproterenol-induced arylalkylamine N- acetyltransferase gene expression in the mouse pineal glandJOURNAL OF PINEAL RESEARCH, Issue 1 2006Mathieu Chansard Abstract:, Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N- acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals. [source] The role of cyclic-AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 6 2006W. Sosroseno Aims:, The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods:, The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results:, Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion:, The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway. [source] Bicarbonate-Induced phosphorylation of p270 protein in mouse sperm by cAMP-Dependent protein kinaseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008Masako Kaneto Abstract Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility. Mol. Reprod. Dev. 75: 1045,1053, 2007. © 2007 Wiley-Liss, Inc. [source] A cyclic adenosine 3,,5,-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Hiroshi Harayama Abstract An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5°C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa. Mol. Reprod. Dev. 1169,1178, 2006. © 2006 Wiley-Liss, Inc. [source] A unique mechanism for cyclic adenosine 3,,5,-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Hiroshi Harayama Abstract A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831,842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-,-cyclodextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes. Mol. Reprod. Dev. 69: 194,204, 2004. © 2004 Wiley-Liss, Inc. [source] | ||||||||||