BACKGROUNDThe 5-hydroxytryptamine7 receptor (5-HT(7) receptor, 5-HT(7)R) plays an important role in the regulation of smooth muscle relaxation and visceral sensation and might be involved in the pathogenesis of the gastrointestinal dyskinesia, abdominal pain and visceral paresthesia in irritable bowel syndrome (IBS). The aim of this study was to investigate the role of the 5-HT(7) receptor in the pathogenesis of IBS.

METHODSA rat model of irritable bowel syndrome with diarrhea (IBS-D) was established by colonic instillation of acetic acid and restraint stress. A rat model with irritable bowel syndrome with constipation (IBS-C) was established by stomach irrigated with 0 - 4 degrees C cool water daily for 14 days. The content and distribution of 5-HT in the brain and gut were examined by immunohistochemistry and the mRNA expression of the 5-HT(7) receptor was determined by fluorescent quantitative reverse transcription polymerase chain reaction. The accumulation of cyclic adenosine monophosphate (cAMP) in all the same tissues was measured by radioimmunity.

RESULTSThe models of IBS were reliable by identification. The immunohistochemistry results showed that there were significantly more 5-HT positive cells in the IBS-D group than in the control group in the hippocampus, hypothalamus, jejunum, ileum, proximate colon and distal colon (P < 0.05), as well as more than were found in the IBS-C group in jejunum and ileum (P < 0.05). There were more 5-HT positive cells in the IBS-C group than in the control hippocampus, hypothalamus, ileum, proximate colon, and distal colon (P < 0.05). Real time-PCR results showed that the expression level of the 5-HT(7) receptor in both the IBS-C and IBS-D groups were enhanced compared with the control group in the hippocampus and hypothalamus (P < 0.05). The expression level of 5-HT(7) receptors in the IBS-C group was notably greater when compared with the controls in the ileum and colon (P < 0.05). The cAMP accumulation in the hippocampus and hypothalamus in both the IBS-C and IBS-D groups was higher than that in the control group (P < 0.01 and P < 0.05). The cAMP accumulation in the IBS-C group was higher than that in the control group in the proximal and distal colon (P < 0.05).

CONCLUSIONSThe increased 5-HT content in the brain and intestine is related to the IBS pathogenesis. The up-regulated expression of the 5-HT(7) receptor in the brain and colon might play an important role in the pathogenesis of IBS-C.

Animals ; Brain ; metabolism ; Cyclic AMP ; metabolism ; Disease Models, Animal ; Immunohistochemistry ; Intestines ; metabolism ; Irritable Bowel Syndrome ; etiology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin ; analysis ; genetics ; physiology ; Serotonin ; analysis

The present study was aimed at examining the regulation of aquaporin (AQP)-2 water channels in the kidney in two-kidney, one clip (2K1C) hypertension. Rats were made 2K1C hypertensive for 6 weeks, and their expression of AQP2 channel proteins was determined in the clipped and contralateral kidneys. To examine the upstream affecting AQP2 channels, adenylyl cyclase activity was also determined. Along with the hypertension, in the clipped kidney, the abundance of AQP2 proteins was significantly decreased in the cortex, outer and inner medulla, while their trafficking remained unaltered. Concomitantly with the reversal of the blood pressure at 24 hours following removal of the clip, the AQP2 abundance also returned to the control level. The arginine vasopressin-evoked generation of cAMP was decreased in the clipped kidney, which again was reversed to the control level following removal of the clip. In contrast, the expression of AQP2 channels as well as the activity of adenylyl cyclase remained unaltered in the contralateral kidney. These results indicate an altered regulation of AQP2 water channels in the clipped kidney in 2K1C hypertension.
Adenylate Cyclase/metabolism ; Animal ; Aquaporins/*analysis ; Blood Pressure ; Cyclic AMP/biosynthesis ; Hypertension, Renovascular/*metabolism ; Kidney/*chemistry ; Male ; Rats ; Rats, Sprague-Dawley

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

OBJECTIVETo study the expression and role of cyclic-AMP response binding protein (CREB) and Bcl-2 in children with acute leukemia.

METHODSNinety-two children with acute leukemia (leukemia group) and 30 children with non-hematologic malignancies (control group) were enrolled. The mRNA and protein expression of CREB and Bcl-2 in bone marrow mononuclear cells were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.

RESULTSThe mRNA and protein expression of CREB and Bcl-2 in the leukemia group was significantly higher than that in the control group (p<0.01). There were no significant differences in the expression of CREB and Bcl-2 between acute lymphoblastic leukemia and acute myeloid leukemia subgroups. At the initial diagnosis, the mRNA and protein expression of CREB and Bcl-2 in children with extramedullary infiltration was higher than that in children without (p<0.05). In the leukemia group, the mRNA and protein expression of CREB and Bcl-2 in the complete remission subgroup was significantly lower than that in the non-complete remission subgroup (p<0.01). High mRNA expression of CREB and Bcl-2 in the leukemia group was positively correlated with peripheral blood leucocyte counts (r=0.62, 0.71 respectively, p<0.05). There was a positive correlation between mRNA and protein expression of CREB and Bcl-2 (r=0.75, 0.68 respectively; p<0.05).

CONCLUSIONSThe expression of CREB and Bcl-2 may be correlated with the pathogenesis and clinical prognosis of childhood leukemia, however, their expression may not be associated with the classification of acute leukemia.

Acute Disease ; Adolescent ; Bone Marrow Cells ; metabolism ; Child ; Child, Preschool ; Cyclic AMP Response Element-Binding Protein ; analysis ; genetics ; Female ; Humans ; Infant ; Leukemia ; metabolism ; Male ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; genetics ; RNA, Messenger ; analysis

OBJECTIVETo observe anti-arrhythmic effect of acupuncture pretreatment in the rat of myocardial ischemia and reperfusion (MIR) and to explore the role of cAMP and Gsa protein in beta-adrenergic receptor signaling.

METHODSMIR was produced by ligation and reperfusion of the left anterior descending coronary artery in the rat. Arrhythmic score, content of cAMP and Gsalpha protein in ischemic myocardium were compared among the normal control (NC), ischemia and reperfusion (IR), electroacupuncture (EA) and EA plus propranolol (EAP) groups.

RESULTSThe arrhythmic score in the IR group at 10 min after reperfusion was higher than the NC group (P < 0.01); in the EA group the score was decreased (P < 0.01 vs the IR group); the score in the EAP group was similar to the IR group, much higher than the EA group (P < 0.01). The similar results for the contents of cAMP and Gsalpha protein were found in the ischemic myocardium. It is suggested that EA pretreatment significantly attenuates the arrhythmic incidence rate and the enhancement of the contents of myocardial cAMP and Gsalpha protein induced by MIR, and the attenuating effect is significantly inhibited by the intraperitoneal pretreatment of propranolol, a specific beta-adrenoceptor antagonist.

CONCLUSIONPretreatment of EA can produce anti-arrhythmic effect in the rat of MIR, which is mediated by the post-receptor signaling pathway of beta-adrenergic receptor.

Acupuncture Therapy ; Animals ; Arrhythmias, Cardiac ; prevention & control ; Calcium ; metabolism ; Cyclic AMP ; analysis ; GTP-Binding Protein alpha Subunits, Gs ; analysis ; Male ; Myocardial Ischemia ; metabolism ; therapy ; Rats ; Rats, Wistar ; Receptors, Adrenergic, beta ; physiology ; Signal Transduction ; physiology

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

BACKGROUNDMost of the studies on traditional Chinese medicine (TCM) 'spleen' deficiency syndrome in the recent 30 years were conducted only on the basis of single functional index, neglecting the study on the pathophysiologic internal relationship between spleen deficiency syndrome and gastric diseases in modern medicine. But it was at the subcellular molecular biological level that we explored the pathophysiologic basis of classification of spleen deficiency in chronic gastritis by detecting the bioactive substances in gastric mucosa nuclei and mitochondria.

METHODSBy means of optical microscope, scanning electron microscope (SEM), transmission electron microscopy (TEM) and histochemical staining, we conducted histopathological, subcellular ultrastructural analysis and nuclei and mitochondrial ultrastructural analysis of gastric mucosa of 188 spleen deficiency patients and of 42 voluntary blood donors. At the same time, bioactive substances were measured by means of X-ray energy dispersive analysis system (EDAX) image analysis system, radioimmunoassay method and chemiluminescence method.

RESULTSThe content of cAMP, superoxide dismutase (SOD), Zn and Cu in gastric mucosa, and the content of Zn and Cu in mitochondria decreased progressively in order of groups: healthy control (HC), spleen Qi deficiency without organic lesion (F-SQD), spleen Yang deficiency without organic lesion (F-SyangD), disease without symptoms group, spleen Qi deficiency with organic lesion (G-SQD), spleen Yang deficiency with organic lesion (G-SyangD), spleen Yin deficiency (SyinD) and spleen deficiency with Qi stagnation (SDQS), chronic spleen deficiency gastritis (CSG) and chronic atrophic gastritis (CAG); decreased in order of HC, intestinal metaplasia (IM)Ia, IMIb, IMIIa and IMIIb, P < 0.05. The content of DNA, Zn and Cu in nuclei progressively increased in order mentioned above, P < 0.05.

CONCLUSIONSThe quantitative changes of gastric mucosal cAMP, SOD, Zn, Cu, of mitochondrial Zn, Cu and of nuclear DNA, Zn and Cu are not only the substance base on which the lesion of gastric mucosa tissue structure occurs, but also the substance base on which spleen deficiency is classified. G-SQD and G-SyangD were more likely to be found in low-grade or middle-grade CSG and CAG, while SyinD and SDQS in middle-grade or high-grade CSG, CAG and IMIIb.

Adult ; Aged ; Chronic Disease ; Cyclic AMP ; analysis ; Female ; Gastric Mucosa ; pathology ; ultrastructure ; Gastritis ; metabolism ; pathology ; Humans ; Lipid Peroxides ; blood ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Splenic Diseases ; classification ; Superoxide Dismutase ; analysis

OBJECTIVETo observe therapeutic effects and mechanisms at different acupoint groups for treatment of the depression rat.

METHODSFifty Wistar rats were randomly divided into a normal group, a model group, a body acupoint group, a scalp acupoint group and a Fluoxetine hydrochloride group, 10 cases in each group. Depression rat model was established by using chronic unpredictable mild stress stimulation combined with lonely breeding for 21 days. EA (2 Hz, 1 mA, 20 min) was given at "Taichong" (LR 3), "Neiguan" (PC 6) and "Zusanli" (ST 36) in the body point group and "Baihui" (GV 20), "Yintang" (EX-HN 3), "Sishencong" (EX-HN 1) in the head point group, once daily, and Fluoxetine hydrochloride was given to the Fluoxetine hydrochloride group by intragastric administration, 2 mg/kg, once daily, for 21 days. Body weight of the rat was recorded, and the sucrose-intake test and forced swimming test were conducted one day before the experiment and on the 7th, 14th and 21st day of the experiment, and the open field test was conducted one day before the experiment and on the 21st day. p-CREB expression in the hippocampus of the rats were observed on the 22nd day.

RESULTSAs compared with the model group, in the head point group and the Fluoxetine group, the crossing and rearing movement times, the relative volume of sucrose-intake and p-CREB expression in the hippocampus increased significantly, and the duration of immovability in forced swimming test shortened significantly (P<0.05). The relative volume of sucrose-intake increased and the duration of immovability in forced swimming test in the body point group significantly shortened (P<0.05), but the crossing and rearing movement times and p-CREB expression in the hippocampus did not insignificantly increase (P>0.05), with no significant differences among the treatment groups (P>0.05).

CONCLUSIONEA at the head acupoints can improve behavioral activities and increase positive neuron number of p-CREB in the hippocampus of the depression rat, and selection of acupoints is of important role in treatment of depression.

Acupuncture Points ; Animals ; Body Weight ; Cyclic AMP Response Element-Binding Protein ; analysis ; Depression ; metabolism ; psychology ; therapy ; Electroacupuncture ; Hippocampus ; chemistry ; Male ; Motor Activity ; Phosphorylation ; Random Allocation ; Rats ; Rats, Wistar ; Swimming

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

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.
Animals ; Bucladesine/pharmacology ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/*metabolism ; DNA-Binding Proteins/chemistry/*genetics/metabolism ; *Gene Expression Regulation ; Humans ; Luciferases/analysis ; Neurons/*metabolism ; PC12 Cells ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins/analysis ; *Response Elements ; Transcription Factors/chemistry/*genetics/metabolism