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1.
To differentiate NPY receptor subtypes, Y1 and Y2, in terms of their impact on feeding behavior, the intact molecule NPY(1–36) and the 3 fragments, NPY(2–36), the Y1 agonist [Leu31,Pro34]NPY, and the Y2 agonist NPY(13–36), were injected (100 pmol/0.3 μl) into the hypothalamic paraventricular nucleus (PVN) of freely feeding rats. A computer-automated data acquisition system was employed in these experiments to permit a detailed analysis of feeding over the 12-h nocturnal cycle, in animals maintained on pure macronutrient diets. The results demonstrate that: 1) NPY(1–36) potentiates feeding behavior, primarily carbohydrate ingestion, by increasing the size and duration of the first meal after injection, rather than by affecting meal number or feeding rate, suggesting that NPY acts through mechanisms of satiety. The potentiation of carbohydrate intake occurs in association with a suppression of protein intake, which is strongest during the second meal after injection and which further increases the proportion of carbohydrate in the diet. No changes in fat ingestion are seen. 2) NPY(2–36), with the N-terminal tyrosine residue deleted, is equally potent to NPY(1–36) in potentiating carbohydrate intake and increasing meal size; however, it is less selective than NPY(1–36), producing an additional, smaller increase in consumption of protein. 3) The stimulatory effect of these peptides on carbohydrate intake and meal size is similarly observed, with somewhat reduced potency, after PVN injection of the selective Y1 agonist [Leu31,Pro34]NPY which, like NPY(1–36), also reduces protein intake. 4) The Y2 receptor agonist, NPY(13–36), causes a decrease in the ingestion of carbohydrate, a smaller decline in protein intake, and a reduction in meal size. It is proposed that hypothalamic Y1 receptors mediate the stimulatory effect of NPY on carbohydrate intake and meal size, while Y2 receptors have the opposite effect of suppressing carbohydrate intake, possibly by altering presynaptic release of monoamines known to influence nutrient ingestion.  相似文献   

2.
To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y2 receptor than for the Y1 receptor. The affinity of [Leu31,Pro34]NPY is 7–60-fold higher for the Y1 receptor when compared with the Y2 subtype. Species selectivity within the Y2 receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y2 subtype (Ki of 0.3 nM) compared with the rabbit and rat Y2 receptor (Ki of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y2 subtype (Ki of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y2 receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.  相似文献   

3.
We have compared the effects of vasoactive intestinal peptide (VIP) and of the VIP-related peptides pituitary adenylate cyclase activating peptide (PACAP) 1–27 and 1–38, helodermin, helospectin I and helospectin II, on the electrically evoked twitches in the isolated vas deferens of the rat. While VIP was virtually without effect, PACAP 1–38 suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC50 value 7.5). The naturally occurring N-terminal fragment PACAP 1–27 was less effective than PACAP 1–38 (Imax values 37.2% suppression compared to 76.5%) and less potent. The C-terminal fragment PACAP 16–38 was virtually inactive. Also helodermin and helospectin I+II suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC50 values 6.9; 7.2; 6.8, respectively). The three peptides produced similar maximum reduction of the twitches (74–80%).

The findings suggest that PACAP, helodermin and helospectin suppress the electrically evoked contractions in the rat vas deferens via receptors distinct from VIP receptors.  相似文献   


4.
We have used an antiserum for immunohistochemistry and RIA/RP-HPLC which recognizes all fragments of N-acetylated endorphin (NacEP). In the rat neurointermediate lobe (N-IL), in addition to the N-acetylated forms of immunoreactive-β-endorphin (ir-βEP) already reported, we have demonstrated NacβEP1–17 as a minor component. In the sheep pituitary processing of βEP is markedly different. In the anterior pituitary (AP), staining was indistinguishable with βEP and NacEP antisera, in contrast with the rat where many fewer AP cells stained with the NacEP antiserum. Secondly, as in the rat, all N-IL cells stained with both antisera; on RP-HPLC, however, the major forms of NacEP in the sheep N-IL were NacβEP1–17 (40%), NacβEP1–27 (25%) and NacβEP1–16 (20%), with NacβEP1–31 (2%) as a minor component. A similar profile was seen on RP-HPLC of sheep AP. These data suggest that (1) patterns of processing in sheep AP are similar to those in N-IL, though the extent of acetylation is less and (2) in the sheep pituitary low molecular weight acetylated fragments predominate, in contrast with the rat.  相似文献   

5.
Self-administration of ethanol and food share many common features and Richter hypothesized that an increase in ethanol consumption would decrease feeding to balance the excess calories contained in the ethanol. Previously, we have shown that individual alcohol consumption correlates with neurotransmitter gene expression, especially in the prefrontal cortex. To test the hypothesis of Richter, we measured hypothalamic gene expression of receptors or neuropeptides of known relevance for the regulation of food intake using qPCR and correlated this to individual ethanol consumption in Wistar rats. For validation, gene expression was first correlated with body weight. We found a correlation of dynorphin, somatostatin, melanocortin-4 receptor and serotonin 5-HT2C with body weight and trends to correlation for CART, thus confirming the established role of the hypothalamus in the regulation of weight. For ethanol consumption, correlations were found for CRH receptors 1 and 2 and vasopressin while strong trends were observed for galanin receptor 1, orexin receptor 1, MCH and adrenoceptor 1B. Therefore, alcohol consumption does seem to involve several hypothalamic systems which also mediate feeding responses and suggests that the hypothalamus, together with the prefrontal cortex, may determine the ‘stopping point’ of an individual.  相似文献   

6.
Post-mortem degradation of brain glutamate decarboxylase   总被引:4,自引:0,他引:4  
The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS–PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD65 and GAD67) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55–57 kDa; no significant amounts of smaller forms were observed. The 55–57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD67 was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79–93 of GAD67), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD65+GAD67), and after 24 h the 55–57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD67 was degraded more rapidly than was GAD65 in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.  相似文献   

7.
The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7–36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7–36)amide and GLP-1(1–36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7–36)amide, GLP-1(1–36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7–36)amide could be identified in the hypothalamus, in amounts that represented 55–94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1–36)amide, representing 0.8–3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7–36)amide (0–0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7–36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27–73% of total GLI, and in five of six specimens only GLP-1(7–36)amide could be identified on HPLC, in amounts representing 13–17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1–36)amide (13% of total GLI) in addition to GLP-1(7–36)amide (31% of total GLI). Acid–ethanol extraction of peptide-free rat plasma with added GLP-1(7–36)amide gave recoveries of 91±SEM 2% in the range 20–200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1±1.1 pmol/l and rose to a maximum of 15.4±3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.  相似文献   

8.
Central injection of Nociceptin/Orphanin FQ (N/OFQ), inhibits the anorectic effect of corticotropin-relasing factor (CRF) and stress in rats. Recently, Urocortin II (Ucn II) and Urocortin III (Ucn III), two selective CRF2 receptor agonists, have been identified. Here, we investigated the effect of intracerebroventricular (ICV) injection of 0.25, 0.75, 1.50 or 3 nmol/rat of Ucn II or Ucn III on food and water intake in food deprived rats. The effect of N/OFQ on Ucn II and UCNIII-induced anorexia was also studied. Results showed a greater inhibition of food consumption by Ucn II than Ucn III. Pretreatment with N/OFQ (0.25–2.0 nmol/rat) did not block the effects of Ucn II and UCNIII. Conversely, injection of N/OFQ (0.25–2.0 nmol/rat) blocked the anorectic effect of CRF (0.1 nmol/rat). These findings suggest that N/OFQ selectively prevent the anorectic effect mediated by activation of the CRF1 receptor system.  相似文献   

9.
Park M  Lin L  Thomas S  Braymer HD  Smith PM  Harrison DH  York DA 《Peptides》2004,25(12):2127-2133
It has been suggested that the F1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin1–7 weakly binds to the rat F1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin1–7 binding to the F1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin1–7 bind to distinct sites on the protein.  相似文献   

10.
125I-labelled GLP-I(7–36)amide was cross-linked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio-labelled band at Mr 66000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 μmol/1 GLP-I(7–36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide-liked protein. The intensity of the radiolabelled protein band was reduced when the incubation with 125I-labelled GLP-I(7–36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP-I(7–36)amide receptor is coupled to the adenylate cyclase system.  相似文献   

11.
Epicuticular wax from Juniperus scopulorum contains hydrocarbons (16%), esters (11%), free acids (1%), nonacosan-10-ol (27%), nonacosane-diols (7%) and estolides (16%). The major hydrocarbon is tritriacontane; the principal esters are C34–C46, mainly octyl and decyl esters of C28-C36 acids; and the diols consist of nonacosane-4,10-diol (57%),5,10-diol (28%), 7,10-diol (11%) and 10,13-diol (4%). Hydrolysis of the estolides gave a mixture of acids, ω-hydroxy acids, , ω-diols, alcohols and hydroxy acids. The hydroxy acids are a new class of C28–C36 acids with the OH attached to either the eighteenth or twentieth carbon from the terminal methyl end; the major component is 13-hydroxydotriacontanoic acid. Syntheses of this acid and of nonacosane-4,10-dione and nonacosane-4,10-diol are described.  相似文献   

12.
R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [125I]-PACAP or [125I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC50 of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC50 in agreement with the pharmacological profile of the VPAC1 receptor subtype: PACAP = VIP > [K15, R16, L27]VIP(1–7)/GRF(8–27) = [R16]ChSn (two VPAC1 agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC2 receptor was inactive at 1 μM. Dose-response curves of VPAC1 agonist molecules (PACAP, VIP, [K15, R16, L27]VIP(1–7)/GRF(8–27), [R16]ChSn) were shifted to the right by the VPAC1 receptor antagonist [AcHis1, D-Phe2, Lys15, Leu17]VIP(3–7)/GRF(8–27), with a Ki of 3 ± 1 nM (n = 3). The presence of VPAC1 receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC1 receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC1 receptors undergoing rapid desensitization in HEL cells.  相似文献   


13.
Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [3H]myoinositol, incubated with additions during 30 min and later [3H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala7]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT1) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala7]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT2) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT2 receptors.  相似文献   

14.
Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu11,D-Trp12,Arg13,Tyr36]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent Kd: 5 × 10−8 M) than [Tyr36]PTHrP(1–36)amide or [Arg11,13,Tyr36]PTHrP(1–36)amide (apparent Kd for both: 2 × 10−9 M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu11,D-Trp12,Arg13,Tyr36,Cys38]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent Kds: 5 × 10−8 M and 8 × 10−8 M), while that of [Nle8,18,Tyr34]bPTH(7–34)amide was more than 10-fold lower (apparent Kd: 2 × 10−6 M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly12.  相似文献   

15.
The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[3H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the Ki value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly Kd values for bladder (+)-[3H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[3H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[3H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in the rat bladder. The ratios of Ki values for (+)-[3H]PN 200–110 to [3H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[3H]PN 200–110, [3H]diltiazem and [3H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The Ki values of propiverine and P-4(N → O) for [3H]diltiazem were significantly smaller than those for (+)-[3H]PN 200–110 and [3H]verapamil. Further, their Ki values for [3H]verapamil were significantly smaller than those for (+)-[3H]PN 200–110. The Ki values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.  相似文献   

16.
Glucose-induced insuline release, glucose-induced rises in intracellular free Ca2+ concentration ([Ca2+]i), and voltage-dependent Ca2+ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca2+]i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca2+]2 rise and, like deprivation of extracellular Ca2+, prevented the glucose-induced rise in [Ca2+]i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca2+ channels was demonstrated directly by measuring Ca2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca2+]1 after membrane depolarization by 45 mMm K+ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.  相似文献   

17.
Nociceptin, the endogenous ligand of the OP4 or ORL1 (opioid receptor-like1) receptor, decreases blood pressure and heart rate in anesthetized rats. Since the OP4 receptor antagonist [Phe1Ψ(CH2-NH)Gly2]-nociceptin(1–13)NH2 possesses an agonistic effect in this model, we examined whether other purported OP4 receptor antagonists, acetyl-RYYRIK-NH2 and naloxone benzoylhydrazone, antagonize the depressant effects of nociceptin. Acetyl-RYYRIK-NH2, like nociceptin and [Phe1Ψ(CH2-NH)Gly2]-nociceptin(1–13)NH2 and unlike naloxone benzoylhydrazone, decreased diastolic blood pressure and heart rate (rank order of potencies: nociceptin ≈ acetyl-RYYRIK-NH2 [Phe1Ψ(CH2-NH)Gly2]-nociceptin(1–13)NH2). The depressant effects were insensitive to the OP1–3 receptor antagonist naloxone but diminished by naloxone benzoylhydrazone. In conclusion, the hypotensive and bradycardic effects of nociceptin in the anesthetized rat are mediated via OP4 receptors, at which acetyl-RYYRIK-NH2 is a highly potent and efficacious agonist.  相似文献   

18.
To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His1,125I-Tyr10,Nle27]hGHRH(1–32)-NH2 increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (Kd = 1.04 ± 0.19 nM, Bmax = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC50) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr1, -Arg2]hGHRH(3–29)-NH2 (3.9 ± 0.58 nM), and [ -Thr7]GHRH(1–29)-NH2 (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.  相似文献   

19.
In a search for novel analogues of β3-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective β3-AR agonist in functional assays using the ferret detrusor (β3-AR), rat uterus (β2-AR), and rat atrium (β1-AR); β3: EC50=7.8 nM, β2: IC50=7,300 nM, β1: EC20=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved β3-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED50=31 μg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e), possess β3-AR agonistic activity in the absence of undesirable β1- or β2-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.  相似文献   

20.
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