Research progress on the role of bile salts in ischemic-type biliary lesion after liver transplantation / 器官移植

Ischemic-type biliary lesion (ITBL) refers to biliary tract injury caused by insufficient blood supply of hepatic artery, which is one of the main factors affecting the long-term survival and quality of life of liver transplant recipients. The incidence of ITBL is associated with cold and warm ischemia, acute and chronic rejection, cytomegalovirus infection and the bile effect, etc. The occurrence of ITBL is a complicated process involving with multiple factors and steps. The therapeutic option of ITBL is extremely limited. A large proportion of ITBL patients should undergo repeated liver transplantation. ITBL has become one of the most critical factors preventing further advancement of liver transplantation. Hence, it is of significance to strengthen prevention and explore more effective modalities. Recent studies have found that toxic injury of bile salts plays a central role in ITBL. Active regulation of bile components, regulation of bile acid-related receptor expression and blockage or activation of bile acid-related signaling pathways probably have potentials in the prevention and treatment of ITBL. In this article, the cytotoxicity of bile salts and the mechanism of bicarbonate umbrella in the incidence and progression of ITBL after liver transplantation were reviewed, aiming to provide reference for the diagnosis and treatment of ITBL.

Research progress in regulating cAMP pathway to prevent and treat chronic obstructive pulmonary disease / 中国药理学通报

Cyclic adenosine monophosphate(cAMP)is a “second messenger” that regulates cell signal transduction. Adenylyl cyclases(ACs)and phosphodiesterases(PDEs)can directly regulate cAMP level in cells and then regulate the downstream signaling pathways. Increasing intracellular cAMP level can inhibit inflammation and enhance smooth muscle relaxation, which is an effective strategy for the prevention and treatment of chronic obstructive pulmonary disease(COPD). This paper briefly summarizes the signaling pathways regulating cAMP and their mechanisms and related drugs in COPD therapy, hoping to provide references for further research and development of new target drugs which regulate cAMP for the prevention and treatment of COPD.

The eukaryotic expression,intracellular location and functions of human CAP1 / 西安交通大学学报(医学版)

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.

Research progress of Gαi/o-coupled receptor-mediated supersensitization of adenylyl cyclase / 药学学报

Persistent activation of most Gαi/o-coupled receptors resulted in enhanced activity of adenylyl cyclase (AC) and subsequent cyclic adenosine monophosphate (cAMP) accumulation within cells, and this phenomenon has generally been referred to as supersensitization of AC. It represents a cellular adaptive response that has been widely believed to be the cause of drug dependence. Supersensitization of AC might have an important impact during the processes of many central nervous system (CNS) disorder diseases, such as schizophrenia and depression, due to altered cell functions. This article provides an overview of the history and present status in our understanding of Gαi/o-coupled receptor-mediated supersensitization of AC, as well as discussion of the problems and future perspective.

Hormonal Sensitivity of Adenylyl Cyclase in the Myocardium, Brain and Testes of 18-month-old Non-Diabetic and Diabetic Rats.

The alterations and abnormalities in hormone-sensitive adenylyl cyclase (AC) system occur at early stages of the type 2 diabetes mellitus (T2DM) and at later stages of the disease they are, according to our view, one of the factors causing T2DM complications. To study the changes in AC system likely to be involved in the development of these complications one needs a very long model of T2DM. Purpose: The aim of this work was the study of AC system in the myocardium, brain and testes of three-, eight- and 18-month-old male rats with experimental T2DM compared with control animals of the same age.

P2 Receptor-mediated Inhibition of Vasopressin-stimulated Fluid Transport and cAMP Responses in AQP2-transfected MDCK Cells

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 ?M) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y2 receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive Gi protein seems to be the underlyihng mechanism.

Altered Regulation of Water Channels and Sodium Transporters in Fasting-induced Polyuria in Rat Kidney / 대한신장학회잡지

PURPOSE: Starvation causes impairment in the urinary concentration ability. However, the molecular basis for the impaired urinary concentration and polyuria remains undefined. We examined the effects of food deprivation on the water handling by the kidney and it's regulatory mechanism. METHODS: Sprague-Dawley rats were used. They were placed in metabolic cages and deprived of food but had free access to water for 24 hours. Control rats had free access to both water and food. Protein expression of aquaporin-2 (AQP2) and Na+-K+-2Cl- cotransporter (NKCC2) was determined in the kidney by Western blot analysis. Protein expression of type VI adenylyl cyclase and prostaglandin E2 synthase (PGES) was determined. Urinary PGE2 excretion was also determined by radioimmunoassay. RESULTS: Food deprivation (FD) resulted in impaired urinary concentration associated with decreased tubular water reabsorption and increased urine output. The expression of AQP2 proteins was significantly decreased in the inner stripe of the outer medulla (ISOM). The expression of NKCC2 was not affected in ISOM. The adenylyl cyclase VI expression was increased in ISOM in FD rats. The protein expression of PGES was decreased in the cortex/OSOM and ISOM. The 24 hr urinary excretion of PGE2 was significantly decreased in FD rats compared with controls. CONCLUSION: These findings indicate that FD-induced urinary concentration defect may related to a reduced abundance of AQP2 in the kidney. It is also suggested that the primary impairment in the pathway to the activation of AQP2 in food deprivation is independent of vasopressin/cAMP or prostaglandin activity.

Dihydroergocriptine increase levels of cAMP and adenylyl cyclase in hippocampus of mice with vascular dementia / 基础医学与临床

Objective To observe the levels of cAMP and adenylyl cyclase(AC) in hippocampus of mice with vascular dementia(VD) and the effect of Dihydroergocriptine(DHE),and to explore the molecular pathogenesis of VD.Methods The mice were subjected for ischemia-reperfusion three times on bilateral common carotid arteries by knots to establish models of VD and the changes of learning and memory were tested on d29/d30 after operation.DHE was administrated to another group of mice,which was taken as treatment group.The cAMP level was evaluated by the radioimmunoassay;AC mRNA positive neurons of hippocampus CA1 area were examined through in-situ hybridization.Results Compared with shamed-operation group,the learning and memory of model group was worse(P

Modulation of Pituitary Somatostatin Receptor Subtype (sst1-5) mRNA Levels by Growth Hormone (GH) -Releasing Hormone in Purified Somatotropes

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH) -releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK (10micrometer). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2> sst5> sst3=sst1> sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.

SR144528 as Inverse Agonist of CB2 Cannabinoid Receptor

It is now well established that several G protein- coupled receptors can signal without agonist stimulation (constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the Gi/o-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.

Functional Role of Serine Residues of Transmembrane Dopamin VII in Signal Transduction of CB2 Cannabinoid Receptor

Using site-directed mutagenesis technique, I have replaced serine 285 and serine 292 with the alanine, and assessed the binding of agonist and signaling such as the inhibition of adenylyl cyclase activity.I have found that serine 292 has an important role in the signal transduction of cannabinoid agonists, HU-210 and CP55940, but not in that of aminoalkylindoles derivatives WIN55,212-2. All mutants express well in protein level determined by western blot using monoclonal antibody HA 11 as compared with the wild type receptor.Interestingly, binding affinity of S285A and S292A mutants with classical cannabinoid agonist HU-243 was somewhat decreased. In signaling assay, the inhibition of adenylyl cyclase by HU-210, CP55940 and WIN55, 212-2 is the same order in both wild type receptor and S285A mutant receptor. However, S292A have been shown that the inhibition curves of adenylyl cyclase activity moved to the right by HU-210 and CP55940, but those of adenylyl cyclase activity did not by aminoalkylindole WIN55,212-2, which is indicating that this residue is closely related to the binding site with HU-210 and CP55940. In addition, serine 292 might take more important role in CB2 receptor and G-protein signaling than serine 285.

PH Domain and Cells Sense of Direction / 生物化学与生物物理进展

The cells sense direction is closely related with the proteins that contain PH (pleckstrin homology) domain. PH domain has been found in about 60 proteins, many of which could activate the sequent events of signal transduction via combining with the related binding sites on the surface of chematactic cel ls. The characteristics of this combining are: a.rapid and transient; b.It is only related to the concentration gradient of surroundings outside cells, whi ch is the base of spatial model; c.The distribution of the binding sites on th e cell membrane changes when the researchers altered the position of the chemoat tractant. This is the base of temporal model. To deeply investigate the effects of all kinds of proteins which contain PH domain on cells sense of direction will greatly promote the research of this field, and hence, has great theoretica l significance.

Altered Renal Expression of Aquaporin-2 Water Channels in Rats with Experimental Two-Kidney, One Clip Hypertension

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.