OBJECTIVETo elucidate the relationship between rat hepatic stellate cells (HSC) and sympathetic neurotransmitter norepinephrine (NE) during liver fibrosis.

METHODSUsing immunofluorescence and RT-PCR, the expressions of a1 and b2-adrenoceptors in activated HSC were detected. Methyl thiazolyl tetrazolium (MTT) was adopted to investigate the effect of NE on the proliferation of HSC. Meanwhile, the expressions of collagen-1, transforming growth factor beta (TGFb) and smooth muscle a-actin (a-SMA) in NE-stimulated HSC were detected by RT-PCR. The contents of NE in HSC were determined by high performance liquid chromatography-electrochemical detector (HPLC-ECD).

RESULTSThe a1 and b2-adrenoceptors were expressed in HSC. NE markedly stimulated the proliferation of HSC in a concentration-dependent manner (F = 140.464, P less than 0.05). NE induced the mRNA expressions of collagen-1, TGFb and a-SMA in HSC (t= -4.160; t= -8.763; t= -17.651, P less than 0.05). HSC were synthesizing and releasing NE, especially when stimulated with platelet-derived growth factor (PDGF) (10 ng/ml) (t= -32.907, P less than 0.05).

CONCLUSIONOur findings show that HSC are direct targets of NE and HSC are hepatic neuroglial cells that produce and respond to sympathetic neurotransmitter norepinephrine, suggesting that interrupting sympathetic nervous system signaling may be useful in the treatment of liver fibrosis.

Actins ; metabolism ; Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Liver Cirrhosis ; Norepinephrine ; pharmacology ; Rats ; Receptors, Adrenergic, alpha-1 ; metabolism ; Receptors, Adrenergic, beta-2 ; metabolism ; Transforming Growth Factor beta ; metabolism

The purpose of the present study was to investigate the effect of chronic intermittent hypobaric hypoxia (CIHH) on &alpha;(1)-adrenergic receptors and the role of alpha(1)-adrenergic receptors in the protection of CIHH against ischemic injury of myocardium. Sixty-six adult male Sprague-Dawley rats were randomly divided into four groups: control group (Con), 14-day CIHH treatment group (CIHH14), 28-day CIHH treatment group (CIHH28) and 42-day CIHH treatment group (CIHH42). CIHH rats were exposed to hypoxia mimicking 5 000 m altitude (p(B)=404 mmHg, p(O(2))=84 mmHg) in a hypobaric chamber, 6 h daily for 14, 28 and 42 d, respectively. Control animals lived in the same environment as CIHH animals except hypoxia exposure. After anesthesia with sodium pentobarbital (3.0-3.5 mL/kg body weight, i.p.), papillary muscle was taken from the right ventricle of rat and perfused with modified Tyrode's solution continuously, at constant temperature (37 °C) and perfusion speed (12 mL/min). Muscle contraction was evoked by electric stimuli. Different concentrations (1x10(-7), 1x10(-6) and 1x10(-5) mol/L) of phenylephrine (PE), an alpha(1)-adrenergic receptor agonist, were applied cumulatively to investigate the effect of PE on the mechanic contraction of right ventricular papillary muscles of rats in Con, CIHH14, CIHH28 and CIHH42 groups. Also, prazosin (1x10(-6) mol/L), an &alpha;(1)-adrenergic receptor antagonist, was used to investigate the role of &alpha;(1)-adrenergic receptor in the protective effect of CIHH on papillary muscle. The results showed: (1) PE increased the maximal isometric tension (P(max)) and maximal velocity of tension development (P(dT/dt)) of muscle contraction in a dose-dependent manner (P<0.05), and the increase of the muscle contraction was much greater in CIHH28 and CIHH42 rats than that in Con rats (P<0.05). Under 1x10(-5) mol/L of PE, the increases of P(max) and P(dT/dt) over the baseline were 51.2% and 44.5% in CIHH28 group, 48.6% and 44.5% in CIHH42 group, and 28.7% and 24.5% in Con group, respectively; (2) The contraction of papillary muscle decreased during simulated ischemia, but the decrease was slighter in CIHH rats than that in Con rats (P<0.05). The decreases in P(max) and P(dT/dt) were 59.6% and 53.6% in CIHH28 group, 60.4% and 49.9% in CIHH42 group, and 74.4% and 64.7% in Con group, respectively; (3) The protective effect of CIHH on ischemic papillary muscle was abolished by prazosin (1x10(-6) mol/L). The results of the present study suggest that CIHH increases the activity of &alpha;(1)-adrenergic receptor, which is possibly one of the mechanisms for the cardioprotection of CIHH.
Altitude ; Animals ; Heart Ventricles ; physiopathology ; Hypoxia ; metabolism ; Male ; Muscle Contraction ; Myocardium ; metabolism ; Papillary Muscles ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1 ; metabolism

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.
Animals ; Diabetes Mellitus, Type 2/drug therapy/*metabolism ; GTP-Binding Protein alpha Subunits/genetics/*metabolism ; Humans ; Insulin-Secreting Cells/metabolism ; Obesity/drug therapy/*metabolism ; Receptor, Melatonin, MT2/genetics/*metabolism ; Receptors, Adrenergic, alpha-1/genetics/*metabolism ; Receptors, Prostaglandin/genetics/*metabolism

OBJECTIVETo investigate the expression and distribution of alpha1-adrenoceptor subtypes in rabbit submandibular gland and the effect of phenylephrine on salivary secretion.

METHODSThe expressions of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein were investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot in rabbit submandibular gland. Immunohistochemical assay was applied to detect the distribution of alpha1A-, alpha1B-, and alpha1D-adrenoceptor and localization of aquaporin 5 in rabbit submandibular gland. Different concentrations of phenylephrine (1 x 10(-8))-(1 x 10(-6)) mol/L were administrated through a polyethylene tube, which was intubated into Wharton's duct of submandibular gland. Heart rate and blood pressure of rabbits were observed during phenylephrine administration. Salivary flow was measured by the length of moist filter paper (35 mm x 5 mm) within 5 min.

RESULTSAlpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein were expressed in rabbit submandibular gland. Three alpha1-adrenoceptor subtypes were widely distributed in the membrane and cytoplasma of both acinar and ductal cells. Phenylephrine (1 x 10(-7) mol/L, 100 microl) stimulated effectively salivary secretion without inducing significant alteration of blood pressure and heart rate in rabbit. Immunohistochemical assay showed that aquaporin 5 was mainly localized in the apical and lateral plasma membrane in both acinar and ductal cells in unstimulated condition, while the expression of aquaporin 5 was increased after administration of phenylephrine.

CONCLUSIONSExpression of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein was existed in rabbit submandibular gland. Phenylephrine safely and effectively promoted salivary secretion when it was administrated through Wharton's duct of submandibular gland. The mechanism of phenylephrine on salivary secretion may involve in the increase of expression of aquaporin 5 in the apical and lateral plasma membrane in rabbit submandibular gland. This study will hopefully lead to a novel strategy for clinical treatment of dysfunction of submandibular gland.

Adrenergic alpha-Agonists ; pharmacology ; Animals ; Aquaporin 5 ; metabolism ; Phenylephrine ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Salivation ; drug effects ; Submandibular Gland ; drug effects ; secretion

New insights in the complex metabolic pathways and its control mechanism for energy homeostasis have refined our understanding of the pathophysiology of obesity. It is now recognized that there are several additional regulatory mechanism such as peripheral signals including leptin, ghrelin, GLP-1 and PYY and cellular signals including uncoupling proteins and beta Adrenergic receptors, which contribute to the regulation of food intake and energy expenditure, respectively. In addition, the function of adipocyte as an endocrine organ in energy homeostasis has been recently emphasized. Recent findings suggest that elevated levels of adipokines, such as leptin, adiponectin, resistin and TNF-alpha, in addition to increased free fatty acid level could be related to the pathophysiology of insulin resistance in obesity. For effective treatments and prevention of obesity, further studies on the circuits of neural and endocrine interactions in the regulation of energy homeostasis are needed.
Adipocytes ; Adipokines ; Adiponectin ; Eating ; Energy Metabolism ; Ghrelin ; Glucagon-Like Peptide 1 ; Homeostasis* ; Insulin Resistance ; Leptin ; Metabolic Networks and Pathways ; Obesity* ; Receptors, Adrenergic, beta ; Resistin ; Tumor Necrosis Factor-alpha

mRNAs of alpha-adrenoceptor (&alpha;-AR) subtypes are found in neurons in dorsal root ganglion (DRG) and change after peripheral nerve injury. In this study, the distribution of &alpha;-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve (CCI). Using immunofluorescence technique, it was found that &alpha;1A-, &alpha;1B-, and &alpha;2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. &alpha;1D- and &alpha;2C-AR was also expressed in all size neurons in normal DRG. However, &alpha;1D-AR was significantly increased and &alpha;2C-AR was decreased in small size neurons 14 days post CCI. &alpha;2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of &alpha;1A- and &alpha;2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of &alpha;-AR subtypes in DRG neurons, and the distribution and levels of expression of &alpha;-AR subtypes change differently after CCI. The up-regulation of &alpha;-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.
Animals ; Cell Size ; Chronic Disease ; Constriction, Pathologic ; Fluorescent Antibody Technique ; Ganglia, Spinal ; metabolism ; pathology ; Male ; Microscopy, Confocal ; Neurons ; metabolism ; pathology ; Pain Measurement ; methods ; Pain Threshold ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1 ; metabolism ; Receptors, Adrenergic, alpha-2 ; metabolism ; Sciatic Nerve ; injuries ; surgery

PURPOSE: To investigate the effects of estrogen on the expression of the alpha1 receptor and nitric oxide synthase (NOS) in rat urethra and bladder after oophorectomy. MATERIALS AND METHODS: Forty-five mature female Sprague-Dawley rats (aged 10-11 weeks, 235-250 g) were randomly assigned to one of three groups: control group, oophorectomy group (Opx), or oophorectomy and estradiol replacement group (Opx+ Est). The degree of expression of alpha1 receptor (alpha1A and D) and NOS (neuronal NOS [nNOS] and endothelial NOS [eNOS]) in bladder and urethral tissues was investigated by using immunohistochemical staining and Western blotting. RESULTS: In the bladder, the expression rates of alpha1 receptor (alpha1A and alpha1D) increased in the Opx group but decreased in the Opx+Est group. These changes were not statistically significant. The alpha1A and alpha1D receptor of the urethra decreased in the Opx group but increased in the Opx+Est group. These changes were not statistically significant. In the bladder and urethra, the expression rates of nNOS and eNOS significantly increased in the Opx group but decreased in the Opx+Est group (p<0.05). CONCLUSIONS: These data suggest that estrogen depletion increases NOS and alpha1 receptor expression in the rat bladder. However, these changes could be restored by estrogen replacement therapy.
Animals ; Collagen/metabolism ; Estradiol/analogs & derivatives/blood/pharmacology ; Estrogen Replacement Therapy/*methods ; Female ; Muscle, Smooth/pathology ; Nitric Oxide Synthase/*metabolism ; Ovariectomy ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1/*metabolism ; Urethra/drug effects/*metabolism/pathology ; Urinary Bladder/drug effects/*metabolism/pathology

Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic protein-1 (BMP-1). In the present study binding between the two proteins was further determined in human embryonic cell 293 (HEK293), a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) and PCP3HA co-transfection group (0.57+/-0.12) were significantly higher than those of the other groups (P<0.001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
Adrenergic alpha-Agonists ; pharmacology ; Bone Morphogenetic Protein 1 ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Cells, Cultured ; Embryo, Mammalian ; Enzyme-Linked Immunosorbent Assay ; Humans ; Kidney ; cytology ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Protein Binding ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

OBJECTIVETo improve the accuracy and sensitivity of cell membrane chromatography (CMC) and evaluate the feasibility of CMC in the study of subtype receptors.

METHODSPlasmids were used to transfer alpha(1B)-AR cDNA into human embryonic kidney 293 (HEK293) cell lines to obtain cell lines stably overexpressing the subtype receptors. HEK293 alpha(1B) cell membrane stationary phase (CMSP) was prepared by immobilizing the cell membrane on silica. The retention time of 9 alpha(1)-adrenoceptor ligands and capacity factors(kappa'(HEK293 alpha1B)) were calculated. The capacity factors of rat liver tissue and primary cultured rat hepatocytes were also calculated for a correlation analysis.

RESULTSThe calculated capacity factors (kappa') were positively correlated to the published pKi values. The affinity rank orders were identical. The longest retention of the 9 alpha(1)-adrenoceptor ligands occurred on CMSP prepared with HEK293 alpha(1B) cell lines, while CMSP obtained from rat liver tissue showed the shortest retention of the ligands.

CONCLUSIONCMC proves practical in the study of the subtype adrenoceptors. The accuracy and sensitivity of CMC can be improved using HEK293 alpha(1B) cell membrane.

Animals ; Cell Membrane ; metabolism ; Chromatography, Affinity ; methods ; DNA, Complementary ; metabolism ; Female ; HEK293 Cells ; Humans ; Kidney ; cytology ; embryology ; Ligands ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1 ; metabolism ; Sensitivity and Specificity

OBJECTIVEDuring the development of hypoxic pulmonary hypertension, the quantity of the protein and mRNA of alpha1-adrenergic receptor (alpha1-adrenergic receptor,alpha1-AR) in the lung tissue increased, while no particular reports were found about the change of the alpha1-AR subtypes during that course. The study aimed to understand the quantity and location of alpha1-AR subtypes mRNA expression in control and hypoxia rats,and the effect of phentolamine in hypoxic pulmonary hypertension.

METHODSFifty-five male Wistar rats were divided randomly into 5 groups: control group (n = 10), hypoxia 2 weeks (n = 13), hypoxia 4 weeks (n = 10), saline control group (n = 10) and hypoxia 4 weeks plus phentolamine group (n = 12). Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) were used to examine the mRNA expression of alpha1-AR subtypes in each group. In situ hybridization was also used to detect the location of the alpha1-AR in lungs of normal rats.

RESULTS(1) The pulmonary artery pressure increased with the extension of hypoxia. (2) The results of RT-PCR showed that the mRNA expression of alpha1A-AR was the most,alpha1B-AR the second and alphaZD-AR was the least in all of the groups. (3) The expression of alpha1A-AR and alpha1B-AR mRNA increased with the extension of hypoxia, and the expressions among groups showed difference. (4) The expression of alpha1D-AR also increased with the extension of hypoxia but no difference was found among groups. (5) No difference was found in mRNA quantity of all three subtypes between phentolamine group and hypoxia saline group. (6) In situ hybridization showed that mRNA of the three alpha1-AR subtypes located mainly in the artery and venous smooth muscle cells and endothelial cells.

CONCLUSIONSThis study suggested that alpha1-AR subtypes worked in the development of hypoxia pulmonary hypertension. More research on alpha1-AR subtypes may help the clinical treatment of pulmonary hypertension.

Adrenergic alpha-Antagonists ; pharmacology ; Animals ; Disease Models, Animal ; Hypertension, Pulmonary ; drug therapy ; etiology ; genetics ; Hypoxia ; complications ; In Situ Hybridization ; Lung ; metabolism ; pathology ; Male ; Phentolamine ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Receptors, Adrenergic, alpha-1 ; classification ; genetics ; Reverse Transcriptase Polymerase Chain Reaction