Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss by modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. This is a review aimed at analyzing the function of fertilization promoting peptide during this process. The possible molecular basis is also discussed.
Acrosome ; drug effects ; Adenylyl Cyclases ; metabolism ; Animals ; Cyclic AMP ; metabolism ; Humans ; Male ; Pyrrolidonecarboxylic Acid ; analogs & derivatives ; Signal Transduction ; drug effects ; Spermatozoa ; drug effects ; physiology ; Thyrotropin-Releasing Hormone ; analogs & derivatives ; pharmacology

AIMTo clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination.

METHODSSpermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined.

RESULTSSupplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C.

CONCLUSIONThese findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

1-Methyl-3-isobutylxanthine ; pharmacology ; Animals ; Bucladesine ; pharmacology ; Cyclic AMP ; physiology ; Cyclic GMP ; analogs & derivatives ; pharmacology ; physiology ; Male ; Papaverine ; pharmacology ; Purinergic P1 Receptor Antagonists ; Sodium Bicarbonate ; pharmacology ; Sperm Agglutination ; drug effects ; physiology ; Sperm Head ; drug effects ; physiology ; Swine ; Theophylline ; analogs & derivatives ; pharmacology

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alpha MSH) inhibits the secretion of interleukin-1 alpha and tumor necrosis factor-alpha from the inflammatory cells, alpha MSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alpha MSH analogues [6His] alpha MSH-ND and [6Asn] alpha MSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC50 of [6His] alpha MSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 0.0045, 1.523 0.707, 0.780 0.405, and 250.320 42.234 nM, respectively, and the EC50 of [6Asn] alpha MSH-ND were 16.8 6.94, 271.8 21.95, 8.0 1.21, and 1132.5 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [6His] alpha MSH-ND and the [6Asn] alpha MSH-ND treated groups (346.0 20.63 g vs. 350.0 13.57 g) were lower than that of the vehicle treated group (375.8 17.31 g, p 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alpha MSH analogues peripherally.
Animal ; Body Weight/drug effects ; Bone and Bones/*drug effects ; CHO Cells ; Cyclic AMP/biosynthesis ; Eating/drug effects ; Hamsters ; Male ; Osteoblasts/drug effects/physiology ; Osteoclasts/drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Corticotropin/physiology ; alpha-MSH/analogs & derivatives/*pharmacology

Epigallocatechin gallate (EGCG) is a principle phenolic antioxidant found in a variety of plants, including green and black tea. The anti-allergic effect of EGCG is unknown. The purpose of this study is to investigate the effects of EGCG on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. For this, the influences of EGCG on the compound 48/80-induced cutaneous reaction were measured in vivo and the effects of EGCG on the compound 48/80-induced mast cell activations were examined in vitro. Results are below: as 1) EGCG significantly inhibited compound 48/80-induced passive cutaneous anaphylaxis, 2) the compound 48/80-induced degranulation, calcium influx and histamine release of rat peritoneal mast cells (RPMCs) were significantly inhibited by the pretreatment with EGCG, and 3) the compound 48/80-mediated inhibition of cAMP level in RPMCs was significantly increased by the pretreatment with EGCG. These results suggested that EGCG, the most abundant polyphenol in green tea, inhibits the compound 48/80-induced mast cell activation and the increase of vascular permeability, and potentially serve as effective therapeutic tools for allergic diseases.
Animals ; Antioxidants/*pharmacology ; Catechin/*analogs & derivatives/pharmacology ; Cyclic AMP/metabolism ; Histamine Release/*drug effects ; Mast Cells/*drug effects/metabolism ; Passive Cutaneous Anaphylaxis/*drug effects ; Rats ; Research Support, Non-U.S. Gov't ; p-Methoxy-N-methylphenethylamine/*antagonists & inhibitors

OBJECTIVETo investigate the potential effects of arsenic trioxide (As(2)O(3)) combined with 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.

METHODSThe RA resistant APL cell lines NB4-R1 and NB4-R2 were used as in vitro models. The effect of As(2)O(3) and/or 8-CPT-cAMP was evaluated according to cellular morphology, cell surface antigen and nitroblue-tetrazolium (NBT) assay. Meanwhile, immunofluorescence analysis and Western blot assay were used to detect the degradation of PML-RAR alpha fusion protein and the change of several key cell cycle regulatory proteins in these cells before and after the treatment.

RESULTSLow dose of As(2)O(3) (0.25 micromol/L) synergized with 8-CPT-cAMP (200 micromol/L) in inducing differentiation of NB4-R1 and NB4-R2 cells, while neither of these two drugs alone could induce differentiation of these cells. In addition, 8-CPT-cAMP was able to inhibit the cell growth by modulating the expression of some important cell cycle regulators and to facilitate the As(2)O(3)-mediated degradation of PML-RAR alpha fusion protein.

CONCLUSIONSAs(2)O(3) combined with 8-CPT-cAMP could induce differentiation of RA-resistant APL cells.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cyclic AMP ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Drug Synergism ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Oxides ; pharmacology ; Thionucleotides ; pharmacology ; Tretinoin ; pharmacology

High level of adenosine A1 receptor-like immunoreactivity has been found in the CA2/CA3a region of adult rat hippocampus, but its roles in the neuronal activity or signal propagation in hippocampus and its intracellular cascade remain to be studied. In this study, we examined the relation between adenosine-3',5'-cyclic monophosphate (cAMP) cascade and suppression of synaptic transmission by endogenous adenosine through adenosine A1 receptor in the CA2 area. In transverse hippocampal slice, maximal electrical stimulation of the hilus region (0.6 mA) only evoked small population spikes (PSs) in the CA2 area (0.5 mV). In the presence of forskolin (20 micromol/L), a direct adenylate cyclase activator, PSs in CA2 were increased to 1.1 mV. When 8-cyclopentyltheophylline (8CPT, 2 micromol/L), an adenosine A1 receptor antagonist, was added in the presence of 20 micromol/L forskolin, PSs with an average amplitude of 4.7 mV were recorded in the CA2 area, much higher than the sum of the amplitude of PSs in the presence of forskolin and 8CPT alone. To test whether this synergistic potentiation results from the additive activation of cAMP cascade, the cAMP content in hippocampal slices was measured with enzyme immunoassay (EIA). Results showed that 8CPT did not increase the cAMP content in CA2 with or without forskolin. Co-application of forskolin and Ro 20-1724, a cAMP-specific phosphodiesterase-IV inhibitor, only increased PSs in CA2 to 1.3 mV but increased cAMP content by 4.4 times. On the other hand, co-application of 8CPT and 1, 9-dideoxyforskolin, a forskolin analog which has no effect on adenylate cyclase, did not mimic the synergistic effect of 8CPT and forskolin on PSs in CA2. These results indicate that up-regulation of adenylate cyclase activity and inhibition of adenosine A1 receptor activity synergistically facilitate the neuronal activity in the CA2 area and the effect of adenosine A1 receptor antagonist is via non-cAMP cascade. These data also suggest that acting on adenosine A1 receptors, endogenous and extragenous adenosine/adenosine A1 agonist(s) inhibit neuronal activity through different pathways.
Adenosine A1 Receptor Antagonists ; Adenylyl Cyclases ; metabolism ; Animals ; Colforsin ; pharmacology ; Cyclic AMP ; physiology ; Drug Synergism ; Hippocampus ; drug effects ; physiology ; Long-Term Potentiation ; drug effects ; Male ; Neurons ; drug effects ; physiology ; Rats ; Rats, Wistar ; Theophylline ; analogs & derivatives ; pharmacology

This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.
Cell Transformation, Neoplastic ; drug effects ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Cyclic AMP ; analogs & derivatives ; pharmacology ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Thionucleotides ; pharmacology ; Tumor Cells, Cultured

The present study was designed to investigate the role of protein kinase A (PKA) and phospholipase A(2) (PLA(2)) in the stimulating effect of adenosine on the basolateral 50 pS K(+) channels in the thick ascending limb (TAL) of the rat kidney. Under the anatomic microscope, the TAL was dissected. The current of 50 pS K(+) channels were recorded by patch clamp technology. The protein expression of phosphorylated PKA and phosphorylated PLA(2) were examined by Western blot. The results showed that cyclohexyladenosine (CHA), an analog of adenosine, increased the 50 pS K(+) channel activity (P < 0.05). In the presence of H8, an antagonist of PKA, CHA did not affect the 50 pS K(+) channel activity. In the presence of AACOCF3 (an antagonist of PLA(2)), CHA did not further increase the 50 pS K(+) channel activity. CHA increased phosphorylation level of PKA, whereas inhibited phosphorylation of PLA(2) in the TAL of the rat kidney (P < 0.01). Furthermore, after blocking the PLA(2) with AACOCF3, CHA still increased the expression of phosphorylated PKA. On the contrary, CHA did not obviously change the expression of phosphorylated PLA(2) after H8 pretreatment. The results suggest that the stimulation of basolateral 50 pS K(+) channels by CHA is mediated by the activation of PKA followed by the inhibition of PLA(2) in the TAL of the rat kidney.
Adenosine ; analogs & derivatives ; pharmacology ; Animals ; Arachidonic Acids ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Kidney ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phospholipases A2 ; metabolism ; Potassium Channels ; metabolism ; Rats ; Signal Transduction

OBJECTIVETo explore the inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation of MRC-5 human fetal lung fibroblasts induced by angiotensin (Ang) II.

METHODSThe study was divided into 2 step: (1) MRC-5 human fetal lung fibroblasts was induced for 48 h at different dose of Ang II and at different time point by 100 nmol/L Ang II. Then the expression of collagen type I and α-smooth muscle actin (α-SMA) were mesaured by western blot. (2) MRC-5 human fetal lung fibroblasts were divided into 4 group: (1) control, (2) Ang II, (3) Ang II+Ac-SDKP, (4) Ang II+8-Me-cAMP (a specific activator of Epac). The α-SMA expression was observed by immnocytochemical stain. The protein expression of collagen type I, α-SMA, serum response factor (SRF), myocardin-related transcription factor (MRTF)-A, exchange protein directly activated by cAMP (Epac) 1, 2 were measured by Westen blot.

RESULTSMyofibroblast differentiation could be induced by Ang II from MRC-5 cells with a dose- and time-dependent manner. The up-regulation of SRF and MRTF-A were observed in MRC-5 cells induced by Ang II and accompanied with collagen I and α-SMA increased. Pre-treatment with 8-Me-cAMP or Ac-SDKP could attenuated all this changes induced by Ang II, and promoted the expression of Epac1.

CONCLUSIONAc-SDKP can inhibit the myofibroblast differentiation of MRC-5 cells induced by Ang II via Epac1 activating.

Actins ; Angiotensin II ; Cell Differentiation ; drug effects ; Collagen ; Collagen Type I ; Cyclic AMP ; analogs & derivatives ; Fetus ; cytology ; Fibroblasts ; cytology ; Guanine Nucleotide Exchange Factors ; Humans ; Lung ; cytology ; Myofibroblasts ; drug effects ; Oligopeptides ; pharmacology ; Serum Response Factor ; Trans-Activators

This study was designed to investigate the effects of cAMP on immune regulation and apoptosis during acute rat cardiac allograft rejection. We found that the production of immune markers such as inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), iNOS expression, and nitric oxide (NO) production, was significantly increased in the blood and transplanted hearts of allograft recipients, but not of isograft controls. These increases were effectively suppressed by the administration of the membrane permeable cAMP analog dibutyryl cAMP (db-cAMP). Administration of db-cAMP reduced allograft-induced elevation of several biochemical markers, such as adhesion molecule expression, iron-nitrosyl complex formation, caspase-3 activation, and apoptotic DNA fragmentation in an animal model. Furthermore, treatment of allograft recipients with db-cAMP prolonged median graft survival to 11 days compared with a median graft survival time of 8 days in saline-treated allograft recipients. These results suggest that db-cAMP exerts a beneficial effect on murine cardiac allograft survival by modulating allogeneic immune response and cytotoxicity.
Animals ; Apoptosis/drug effects ; Caspase 3/metabolism ; Cyclic AMP/analogs & derivatives/*pharmacology/*therapeutic use ; Electron Spin Resonance Spectroscopy ; Graft Rejection/*drug therapy ; Graft Survival/*drug effects ; Heart Transplantation/*adverse effects ; Interleukin-1beta/metabolism ; Interleukin-6/metabolism ; Male ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type II/genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha/metabolism