中文网站正在持续更新中,请密切关注我们康肽生物的最新动态,或点击访问右上角的英文官方网站 www.phoenixpeptide.com
PHOENIX PHARMACEUTICALS, INC. TOP HOME PAGE
Top Catalog English Version | My Account | 联系我们 | China



 多肽



 标记多肽 



 多肽激素文库



 抗体 



 免疫试剂盒 



 生物标志物阵列 



 多肽样品检测



 自定义肽链合成及GMP



 产品目录索取



 样品准备



 提问和解答


Prolactin-releasing Peptide (PrRP)

Prolactin Releasing Peptides (PrRP) Related Products

 


Alternative role for prolactin-releasing peptide in the regulation of food intake.
Catherine B. Lawrence, et al. Nature Neuroscience 3, 645 - 646 (2000)

 


PrRP affects food intake and core body temperature in free-fed animals.
(a) Cumulative food intake was significantly reduced after 4 nmol PrRP (i.c.v.; * p < 0.05 versus vehicle). (b) Temperature is expressed as change in core body temperature from baseline (time zero) and was measured by remote telemetry. 2 nmol or 4 nmol PrRP caused a hypothermic response that reached a nadir at 30 min and then returned to above control levels at 2 h after injection. Rat PrRP-31 (Bachem, Saffron Walden, UK) was injected through indwelling catheters into the lateral ventricle of conscious, free-moving, male Sprague-Dawley rats (n = 6-7 per group). Food and water (see text) intake data were analyzed using repeated measures ANOVA with Scheffe's post-hoc comparisons. Otherwise, analyses used a one-way ANOVA with Tukey post-hoc comparisons.
Catherine B. Lawrence, et al. Nature Neuroscience 3, 645 - 646 (2000)

 


PrRP reduces fast-induced re-feeding.
Animals were allowed to re-feed after a 24-h fast. (a) 4 nmol PrRP significantly reduced the initial re-feeding response, though food intake was normalized in PrRP-treated animals during the 24 h after injection. (b)Although core body temperature is strongly affected by fast-induced re-feeding, the change in body temperature was consistently above that of controls for a period of several hours. Statistics involved repeated - measures ANOVA and t-tests. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus vehicle-treated group.
Catherine B. Lawrence, et al. Nature Neuroscience 3, 645 - 646 (2000)

 


(a) Total, non-specific and specific binding of [125I]-PrRP-20 to membranes from HEK293 cells expressing GPR10 receptors with increasing radioligand concentration. Data represents a single experiment (each point determined in quadruplicate), which was replicated six times with similar results. (b) Scatchard transformation of the data from (a).
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 


(a) Time course for association of [125I]-PrRP-20 binding to HEK293-GPR10 receptor expressing membranes. Data represents a single experiment, which was replicated five times with similar results. The inset shows the data transformed as a semi-log plot (correlation coefficient r=0.98), where Bt is the specific binding at time t and Be is the specific binding measured at equilibrium. (b) Time course for the dissociation of [125I]-PrRP-20 binding from HEK293-GPR10 receptor expressing membranes. Data represents a single experiment, which was replicated three times with similar results. The inset shows the data transformed as semi-log plot, where Bt is the specific binding at time t and Be is the specific binding measured at equilibrium.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 


Competition for [125I]-PrRP-20 binding to HEK293-GPR10 expressing membranes by rat and human PrRP-20 and PrRP-31. [125I]-PrRP-20 (0.2 nM) was incubated in the presence of increasing concentrations of the compounds. Data are the mean of at least three independent experiments; vertical lines show s.e.mean.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 


Concentration dependent stimulation of Ca2+ mobilization by human PrRP-20, human PrRP-31, rat PrRP-20 and rat PrRP-31 in HEK293 cells expressing GPR10. Data shown are the mean of six experiments; vertical lines show s.e.mean.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 


The distribution of 125I-PrRP binding in rat peripheral tissues (A) and the central nervous system (B) in the presence or absence of 10 nM hPrRP. Rat tissue membranes (100 g membrane protein) were prepared and binding assays using 125I-hPrRP (750 Bq, 30 pM) performed as described in the Methods. Binding is presented as fmols of specific 125I-hPrRP binding per mg protein. All binding assays were performed in triplicate in the presence and absence of 1 M hPrRP to calculate specific binding (labelled total binding, filled bars). Specific binding was also measured in the presence of 10 nM hPrRP (open bars). Each time point is the means.e.mean of at least three separate membrane preparations. WAT is white adipose tissue; S.I. is small intestine; medulla is medulla oblongata; cortex is cerebral cortex.
Fumitoshi Satoh, et al. British Journal of Pharmacology (2000) 129, 1787-1793

 


Competition for 125I-PrRP binding by hPrRP in rat hypothalamic (A), pituitary (B), heart (C) and soleus muscle (D) membranes. Rat tissue membranes (100 g membrane protein) were prepared and binding assays using 125I-hPrRP (750 Bq, 30 pM) performed as described in the Methods. Non-specific binding was measured in the presence of 1 M hPrRP. Binding is shown as a percentage of the maximal specific binding in the absence of unlabelled PrRP. All binding assays were performed in triplicate and the curves shown are meanss.e.mean of five (hypothalamus), four (heart and soleus) and three (pituitary) separate experiments.
Fumitoshi Satoh, et al. British Journal of Pharmacology (2000) 129, 1787-1793

 


Amino acid sequences of short and long forms of prolactin releasing peptide (PrRP) in rat and human.Langmead C.L. et al.
British Journal of Pharmacology (2000) 131, 683-688

 


Membrane preparation

Human embryonic kidney cells (HEK293) stably transfected with GPR10 were harvested with PBS, pelleted and stored at -80C until further use. Membranes were prepared using a modification of the method of Miyamoto et al. (1994); all procedures were carried out at 4C. In brief, cells were washed in 30 vols (w v-1) of PBS with 0.2 mM EDTA. The suspension was homogenized using an Ultra-Turrax homogenizer and the subsequent homogenates centrifuged at 39,000g for 15 min. The resultant pellets were resuspended in 30 volumes of buffer containing 10 mM Na2CO3, 1 mM EDTA, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), 1 g ml-1 pepstatin and 1CompleteTM serine and cysteine protease inhibitor tablet 250 ml-1 (pH 7.4). The suspension was then homogenized and centrifuged at 1000g for 10 min, the supernatant decanted and centrifuged at 48,000g for 20 min. The resultant pellets were resuspended in buffer containing 20 mM Tris-HCl, 0.25 M sucrose, 2 mM EDTA, 0.5 mM PMSF, 1 g ml-1 pepstatin and 1CompleteTM serine and cysteine protease inhibitor tablet 250 ml-1 (pH 7.4) to a volume of approximately 48106 cells ml-1 and stored at -80C until used.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 

[125I]-PrRP-20 binding assays

HEK293-GPR10 receptor expressing cell membranes were incubated with [125I]-PrRP-20 in buffer containing 20 mM Tris-HCl, 5 mM Mg-Acetate, 2 mM EGTA, 0.5 mM PMSF, 1 g ml-1 pepstatin and 1CompleteTM serine and cysteine protease inhibitor tablet 250 ml-1 and 0.1% (w v-1) BSA (pH 7.4) at 25C for 90 min. The total assay volume was 0.5 ml. The reaction was terminated by rapid filtration through Whatman GF/B glass fibre filters, followed by rapid washing of the filters with 51 ml aliquots of ice cold buffer containing 50 mM Tris-HCl, 10 mM MgCl2 (pH 7.4). Bound radioactivity was determined by gamma counting. Non-specific binding was defined as that remaining in the presence of 0.1 M rat PrRP-31. Saturation studies were carried out by incubating membranes (4 g protein well-1) with a range of concentrations of [125I]-PrRP-20 (0.01-5 nM). Specific binding data was analysed using the program Radlig (Biosoft) to provide estimates of KD and Bmax values. Protein content was assayed using the Bradford method (Bradford, 1976) using bovine serum albumin as a standard. Association kinetic studies were performed by measuring specific binding of [125I]-PrRP-20 (0.2 nM) at 0.5-90 min after addition of membranes (2 g protein well-1). For dissociation studies, membranes were pre-incubated with [125I]-PrRP-20 (0.2 nM) for 90 min. Specific binding was then measured at 5-200 min after the addition of 0.1 M PrRP-31. Kinetic data was analysed by GraFit (Erithacus Software) to provide estimates of Kon and Koff values. Competition studies were performed by incubating cell membranes (2 g protein well-1) with [125I]-PrRP-20 (0.2 nM) and a range of concentrations of the test compound. Competition curves were analysed by non-linear least-squares fitting to a four parameter logistic equation by Microsoft Excel in order to determine IC50 values (Bowen & Jerman, 1995). Ki values were then derived from the IC50 values using a nominal KD value of 0.1 nM (which takes into account binding to both high and low affinity sites obtained from saturation studies) (Cheng & Prussoff, 1973). Results are given as means (s.e.mean) of at least three independent experiments.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

 

Calcium mobilization assays
Intracellular calcium was monitored using the fluorescent dye Fluo 4AM in a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, U.K.). HEK293-GPR10 cells were cultured in poly-D-lysine coated 96-well microtitre plates 24 h before use at as a seeding density of 52,000 cells well-1. Prior to assay on FLIPR, cells were incubated with Fluo 4AM (1 M) for 60 min at 37C in Hank's buffered saline solution containing 0.1% BSA and 2.5 mM probenicid. Extracellular dye was then removed by washing three times with 150 l Hank's buffered saline solution containing 2.5 mM probenicid without BSA. Compounds were tested for agonist activity in FLIPR by adding 40 l of test solution to a plate volume of 120 l at 37C. Peak stimulation (minus basal) was plotted versus concentration of test compound and iteratively curve fitted using a four parameter logistic equation (Grafit, Erithacus Software) to assess agonist potency and maximal response.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688
 
 
 

 

1. Lawrence CB, Celsi F, Brennand J, Luckman SM. Alternative role for prolactin-releasing peptide in the regulation of food intake.  Nat Neurosci 2000 Jul;3(7):645-646   School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT UK.
1. Prolactin-releasing peptide (PrRP) is a peptide ligand for the human orphan G-protein-coupled receptor hGR3/GPR10 and causes the secretion of prolactin from anterior pituitary cells. However, the lack of immunoreactive staining for PrRP in the external layer of the median eminence seems to rule out this peptide as a classical hypophysiotropic hormone and, furthermore, PrRP is less effective than another inducer of prolactin secretion, thyrotropin-releasing hormone, both in vitro and in vivo. Here we show a reduction in the expression of PrRP mRNA during lactation and fasting and an acute effect of PrRP on food intake and body weight, supporting the hypothesis of an alternative role for the peptide.

 

2. Samson WK, Resch ZT, Murphy TC. A novel action of the newly described prolactin-releasing peptides: cardiovascular regulation.  Brain Res 2000 Mar 6;858(1):19-25.

 

3. Satoh F, Smith DM, Gardiner JV, Mahmoodi M, Murphy KG, Ghatei MA, Bloom SR
Characterization and distribution of prolactin releasing peptide (PrRP) binding sites in the rat--evidence for a novel binding site subtype in cardiac and skeletal muscle. Br J Pharmacol 2000 Apr;129(8):1787-93
Endocrine Unit of the Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, W12 ONN.
Prolactin releasing peptide (PrRP) was recently purified from bovine hypothalamus and binds to the orphan receptor, UHR-1. We examined the distribution and kinetics of (125)I-PrRP binding in rat tissues together with molecular characterization by chemical cross-linking and Northern blotting. In this study (125)I-PrRP binding showed specificity and rapid association and dissociation. Specific binding was found in membranes from rat tissues including brain (hypothalamus, medulla oblongata and cerebellum), pituitary, heart, soleus muscle, adipose tissue, kidney, adrenal gland, testis and small intestine. In hypothalamus, pituitary, heart and soleus competition analysis indicated only one class of binding site in each tissue. Binding affinity for PrRP (IC(50)) and binding site density (B(max)) respectively were 5.2+/-0.9 nM and 674+/-97 fmol mg protein(-1) in hypothalamus (n = 5), 1.4+/-0.6 nM and 541+/-126 fmol mg protein(-1) in pituitary (n = 3), 6.6+/-0.7 nM and 628+/-74 fmol mg protein(-1) in heart (n = 4) and 9.8+/-0.9 nM and 677+/-121 Soun mg protein(-1) in soleus muscle (n = 4). Analysis of (125)I-PrRP-binding site complexes by chemical cross-linking showed a binding site M(r) of 69,000 in hypothalamus and 41,000 in heart and soleus. Northern analysis of polyA(+) RNA from hypothalamus showed a 4.2 kb band as expected for UHR-1, but heart and soleus showed a 4.8 kb band. Taken together these results indicate that there may be different subtypes of PrRP binding sites in rat tissues which may differ from UHR-1.

 

4. Matsumoto H, Murakami Y, Horikoshi Y, Noguchi J, Habata Y, Kitada C, Hinuma S, Onda H, Fujino M. Distribution and characterization of immunoreactive prolactin-releasing peptide (PrRP) in rat tissue and plasma. Biochem Biophys Res Commun 1999 Apr 13;257(2):264-8
We established a sensitive and specific two-site enzyme immunoassay (EIA) for prolactin-releasing peptide (PrRP) using two region-specific monoclonal antibodies. We investigated the tissue distribution and the plasma concentration of immunoreactive (ir-) PrRP in rats using this assay. Ir-PrRP was widely distributed in the central nervous system and pituitary gland. The highest concentration of ir-PrRP was found in the hypothalamus. In peripheral tissues, appreciable levels of ir-PrRP were found only in the adrenal gland. The mean plasma concentration of ir-PrRP was 0.13 +/- 0.01 fmol/ml (mean +/- SEM). In reverse-phase and gel-filtration high performance liquid chromatography, hypothalamic ir-PrRP eluted at a position identical to that of PrRP31 and PrRP20. On the other hand, ir-PrRP from the adrenal gland and plasma eluted only at the position of synthetic PrRP31, indicating that molecular forms of ir-PrRP in vivo differed among tissues. Copyright 1999 Academic Press.

The prolactin-releasing peptide receptor (GPR10) regulates body weight homeostasis in mice.
To identify new drug targets for the treatment of obesity, we employed a degenerate reverse transcriptasepolymerase chain reaction technique to isolate novel members of the G-protein coupled receptor superfamily from mouse hypothalamus. One of our clones was found to encode a protein with 90% amino acid identity to human GPR10, which was previously identified as the receptor for prolactin-releasing peptide (PrRP) and has been implicated in lactation, the regulation of food intake and other physiological functions. To investigate the role of GPR10 in food intake and body weight homeostasis, we generated mice carrying a targeted deletion of the GPR10 gene. First, using these knockout animals, we confirmed that GPR10 is the principle receptor for PrRP in the mouse hypothalamus because deletion of GPR10 completely abolished PrRP binding to isolated hypothalamic cell membranes. Second, we investigated the effect of normal and high-fat diets on energy intake, body weight, and glucose homeostasis in wild-type and GPR10 knockout mice. After fasting and refeeding, food intake in knockout animals was unchanged relative to control littermates. However, beginning at 16 wk of age on a normal diet, knockout mice became hyperphagic, obese, and showed significant increases in body fat and the levels of leptin and insulin, as well as decreased glucose tolerance. This metabolic profile was similar to the effect of a high-fat diet on wild-type animals. Our findings provide direct evidence that GPR10 is the receptor for PrRP and that it is involved in the regulation of energy balance in mice. GPR10 knockout mice will also prove useful for investigating other proposed activities for PrRP.
Gu W, et al. J Mol Neurosci. 2004;22(1-2):93-103

PrRP Related Products

%Prolactin-Releasing Peptide%


分类搜索
关键字搜索
按字母搜索
A B C D E F G H I J K L M N
O P Q R S T U V W X Y Z

Copyright 2024 PHOENIX BIOTECH