Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)₂D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l^-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)₂D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.
Adenosine Triphosphate/metabolism* ; Adult ; Calcium/metabolism* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Humans ; Male ; Semen/metabolism* ; Semen Analysis ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Vitamin D/pharmacology* ; Vitamin D Deficiency/blood* ; Wit and Humor as Topic ; Young Adult

OBJECTIVEThis study examined the effects of prenatal application of taurine on mRNA expression of protein kinase A cAMP response element binding protein (PKA-CREB) signal pathway and glial cell line derived neurotrophic factor (GDNF) in fetal rat brains of intrauterine growth restriction (IUGR).

METHODSPregnant rats were randomly divided into 4 groups: normal control, IUGR model, low dose (100 mg/kg x d) and high dose (300 mg/kg x d) taurine treatment IUGR (n = 5 each). IUGR was induced by food restriction throughout pregnancy. PKA, CREB and GDNF mRNA expression in brains of newborn rats was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSPKA, CREB and GDNF mRNA expression in the IUGR model group was significantly higher than that in the normal control group (p<0.05). Compared with the IUGR model group, mRNA expression of PKA and CREB in both the low dose and high dose taurine treatment groups increased significantly (p<0.05); GDNF mRNA expression in the high dose taurine treatment group also increased significantly (p<0.01).

CONCLUSIONSTaurine can increase mRNA expression of PKA, CREB and GDNF in fetal rat brains of IUGR. This suggests that prenatal application of taurine may increase neurogenesis of the central nervous system and endogenous secretion of neurotrophic factors, thus providing neuroprotective effects.

Animals ; Brain ; drug effects ; Cyclic AMP Response Element-Binding Protein ; genetics ; physiology ; Cyclic AMP-Dependent Protein Kinases ; genetics ; physiology ; Female ; Fetal Growth Retardation ; metabolism ; Fetus ; drug effects ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Male ; Pregnancy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Taurine ; pharmacology

BACKGROUNDThe effect of chronic stress on cognitive functions has been one of the hot topic in neuroscience. But there has been much controversy over its mechanism. Such single stressor applied in the past could not simulate complicated living circumstances that people confronted with. The aim of this study was to investigate the effects of chronic multiple-stress on learning and memory as well as on the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), calmodulin (CaM) mRNA, and cAMP-response element binding protein (CREB) mRNA in the hippocampus of rats.

METHODSThe rats were divided randomly into stressed and control groups. The stressed group was given chronic multiple-stress for 6 weeks to set up a chronic multiple-stressed model. The rats' performance of spatial learning and memory was tested using Morris Water Maze (MWM) and Y-maze. Meanwhile, the expressions of CaMKII, CaM mRNA and CREB mRNA of rats' hippocampus were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats by electron microscopy.

RESULTSAfter exposure to chronic multiple-stress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P < 0.05, P < 0.01). The width of synaptic cleft was smaller and the thickness of PSD was larger in the hippocampal CA3 region of the stressed group than in that of the control group (P < 0.01). The CaMK II immunostaining of the stressed group was stronger than that of the control group in the stratum radiatum and oriens of the hippocampal CA1 and CA3, especially in the stratum oriens. Quantitative analysis indicated that the expression of CaMK II, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was higher than that of the control group (P < 0.05, P < 0.01).

CONCLUSIONSThe capacity of learning and memory can be enhanced after chronic multiple-stress. The increased levels of CaMK II, CaM mRNA, and CREB mRNA may contribute to the enhancing effect of chronic multiple-stress on learning and memory.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; Calmodulin ; genetics ; Chronic Disease ; Cyclic AMP Response Element-Binding Protein ; genetics ; Hippocampus ; metabolism ; ultrastructure ; Learning ; Male ; Memory ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Stress, Physiological ; metabolism ; psychology ; Synapses ; ultrastructure

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.
Animals ; Carbazoles/pharmacology ; Cell Line ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; DNA-Binding Protein, Cyclic AMP-Responsive/metabolism ; DNA-Binding Proteins/metabolism ; Gangliosides/pharmacology/*physiology ; Gene Expression Regulation ; Indoles/pharmacology ; Interleukin-1/genetics ; Isoquinolines/pharmacology ; Mice ; Microglia/drug effects/*enzymology/*immunology ; NF-kappa B/metabolism ; Nitric-Oxide Synthase/genetics ; Plasminogen/pharmacology/*physiology ; Pyrroles/pharmacology ; RNA, Messenger/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Sulfonamides/pharmacology ; Tumor Necrosis Factor-alpha/genetics

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.
Animals ; Aorta, Thoracic/cytology ; Blotting, Western ; Cattle ; Cell Culture Techniques ; Cell Extracts ; Cells, Cultured ; Comparative Study ; Cyclic AMP-Dependent Protein Kinases/analysis/*metabolism ; Endothelium, Vascular/cytology/*enzymology/*metabolism ; Nitric Oxide Synthase Type III/analysis/*metabolism ; Phosphorylation ; Precipitin Tests ; Research Support, Non-U.S. Gov't ; Stress, Mechanical ; Time Factors

We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolatioin and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.
Sequence Homology, Amino Acid ; Sequence Analysis, DNA ; Rats ; Protein Isoforms/genetics ; Phosphorylation ; Phosphoproteins/genetics/*metabolism ; Molecular Sequence Data ; Mice ; Male ; Humans ; Gene Expression Profiling ; Female ; DNA, Complementary/chemistry/genetics ; Cyclic AMP-Dependent Protein Kinases/*physiology ; Cloning, Molecular ; Cell Line ; Base Sequence ; Animals ; Amino Acid Sequence ; Adult

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.
Animal ; Blotting, Western ; Bucladesine/pharmacology ; Cholera Toxin/pharmacology* ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Forskolin/pharmacology ; GTP-Binding Proteins/genetics* ; GTP-Binding Proteins/biosynthesis ; Gene Expression Regulation* ; Glioma ; Membrane Proteins/analysis ; RNA, Messenger/metabolism ; RNA, Messenger/genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction