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

Human embryonic kidney cells (HEK293) stably transfected with GPR10 were harvested with PBS, pelleted and stored at -80°C until further use. Membranes were prepared using a modification of the method of Miyamoto et al. (1994); all procedures were carried out at 4°C. 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 -80°C until used.
Langmead C.L. et al. British Journal of Pharmacology (2000) 131, 683-688

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 25°C 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