Characterization of Neuropeptide Y Receptors Mediating Contraction, Potentiation and Inhibition of Relaxation

Nilsson T, You J, Sun X, Hedner T, Edvinsson L. Characterization of neuropeptide Y receptors mediating contraction potentiation and inhibition of relaxation.

In addition to its direct vasoconstrictive effect, neuropeptide Y (NPY) potentiates noradrenaline- (NA) induced contraction and inhibits acetylcholine- (ACh) induced relaxation: The aim of the present study was to elucidate the NPY receptor subtypes responsible for mediating these three responses. NPY, peptide YY (PYY) and pro³⁴NPY (a NPY Y₁ receptor agonist) induced equipotent and equally strong concentration-dependent contractions of guinea pig basilar arteries. NPY_13-36 (a NPY Y₂ receptor agonist), however, caused only weak contraction with significantly lower potency. The NPY-induced contraction was significantly inhibited by the selective NPY Y₁ receptor antagonist BIBP3226 (1 μM). NPY, PYY and pro³⁴NNPY but not NPY_13-36 significantly potentiated the NA-induced contraction in guinea pig mesenteric arteries. The potentiation was significantly inhibited by BIBP3226 (IpM). In precontracted guinea pig basilar arteries, ACh induced a concentration-dependent relaxation which was significantly inhibited by NPY, PYY and NPY_13-36 but not by pro³⁴NPY. BIBP3226 had no significant effect on the NPY-induced inhibition of the relaxation. These results suggests that the NPY Y, receptors mediate the direct contraction and the potentiation of the NA-induced contraction but not the inhibition of the ACh-induced relaxation. This effect seems to be mediated by another NPY receptor subtype, presumably by the Y₂ receptor, as judged from the agonist potency order.     

钙离子在大鼠结肠平滑肌运动中作用机制的研究

目的 应用束缚应激大鼠实验模型,研究离体结肠平滑肌的收缩运动及其影响因素,探讨鸟苷素在结肠运动中的作用。方法 建立束缚应激大鼠动物模型,制备离体结肠平滑肌环行肌及纵行肌肌条,应用张力换能器,测定其肌张力。应用放射配基法测定结肠组织及血浆中鸟苷素含量。结果 束缚应激刺激可诱发大鼠排便增加,该动物模型是较好的模拟人ＩＢＳ的实验动物模型。束缚应激大鼠离体结肠平滑肌的张力升高,对Ｋ＾＋、Ｃａ＾２＋、乙酰胆     

Gq-linked NK(2) receptors mediate neurally induced contraction of human sigmoid circular smooth muscle

BACKGROUND & AIMS: Because tachykinins have been identified as neurotransmitters in the guinea pig colon and human ileum, we examined a possible role of tachykinin receptors and neurokinin (NK) A in neurally induced contraction of human sigmoid colon circular muscle. METHODS: Muscle strips were stimulated electrically for 10 seconds. Single cells were isolated by enzymatic digestion and permeabilized by saponin. [(35)S]GTPgammaS binding was assayed with or without NKA for 5 minutes. Intracellular Ca(2+) was measured using Fura 2. RESULTS: In the presence of 100 micromol/L L-NNA, 100 micromol/L atropine did not affect electrical field stimulation (EFS)-induced contraction. A peptide NK(2)-receptor antagonist (NK-2ra) but not an NK(1) antagonist FK888 (1 micromol/L) eliminated EFS-induced contraction. NKA-induced contraction in muscle strips and single cells was virtually abolished by NK-2ra, but not by FK888. In permeabilized cells, contraction was blocked by Gq-protein antibodies, but not by other G-protein antibodies, suggesting that NKA activates Gq, which was confirmed by a [(35)S]GTPgammaS binding assay. NKA-induced contraction and increase in cytosolic Ca(2+) were abolished by depletion of intracellular Ca(2+) stores. CONCLUSIONS: Tachykinins may be the main excitatory neurotransmitters in human sigmoid circular muscle. NKA activates Gq-linked NK(2) receptors, which cause Ca(2+) release, followed by contraction.     

Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice

Neuropeptide Y (NPY) increases food intake through the action of hypothalamic NPY receptors. At least six subtypes of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) receptors have been identified in mice. Although the involvement of Y1 and Y5 receptors in feeding regulation has been suggested, the relative importance of each of these NPY receptors and the participation of a novel feeding receptor are still unclear. To address this issue, we generated a Y1 receptor-deficient (Y1-/-) and a Y5 receptor-deficient (Y5-/-) mouse line in which we directly compared the orexigenic effects of NPY and its analogs after intracerebroventricular (icv) administration. The icv NPY-induced food intake was remarkably reduced in Y1-/- mice, but was not significantly altered by inactivation of the Y5 receptor. The Y1 receptor therefore plays a dominant role in NPY-induced feeding. Stimulation of feeding by moderately selective Y5 agonists [PYY-(3-36), human PP, and bovine PP] was reduced in Y5-/- mice, although food intake did not decrease to vehicle control levels. These results indicate that the Y5 receptor functions as one of the feeding receptors. In addition, the finding that Y5-preferring agonists still induce food intake in Y5-/- mice suggests a role for another NPY receptor(s), including the possibility of novel NPY receptors. Surprisingly, despite the limited efficacy of PYY-(3-36) and PPs at the Y1 receptor, food consumption induced by these agonists was significantly diminished in Y1-/- mice compared with that in wild-type controls. These observations suggest that the feeding stimulation induced by NPY and its analogs may be directly or indirectly modulated by the action of the Y1 receptor. We conclude that multiple NPY receptors, possibly including the novel feeding receptor, are involved in the feeding response evoked by NPY and its analogs. Among them, the Y1 receptor plays a key role in NPY-induced feeding in mice.     

Characterization of the cloned atlantic cod neuropeptide Y-Yb receptor: peptide-binding requirements distinct from known mammalian Y receptors.

Five members of the neuropeptide Y (NPY) receptor family have been cloned in mammals. The recently cloned NPY receptor in the Atlantic cod seems to be distinct from the mammalian subtypes as it has only 50% identity to Y1, Y4, and y6 and only 30% to Y2 and Y5. In most of the other families of G-protein-coupled receptors, species homologues have 65-90% identity between fishes and mammals. The functional expression and detailed pharmacological characterization of this cod NPY receptor, designated Yb, is reported. Membranes of cells transiently transfected with cod Yb showed saturable [(125)I]PYY binding with a K(d) of 45 pM. The pharmacological profile is similar to those of both the zebrafish Yb and Yc receptors and distinct from those of the mammalian NPY receptors. In competition experiments the cod Yb receptor had the following rank order of potencies: porcine PYY = porcine NPY = p[Leu(31), Pro(34)]NPY > zebrafish PYY > zebrafish NPY > NPY2-36 = NPY3-36 > NPY18-36 > bovine PP = [D-Trp(32)]NPY > BIBP3226. This is in sharp contrast to the high selectivity of BIBP3226 for the Y1 receptor from all mammalian species. Together with the low amino acid identity of cod Yb with the mammalian Y1, Y4, and y6 receptors, this is further support for the notion that fish Yb constitutes a distinct NPY receptor subtype.     

Brain peptide YY receptors: highly conserved characteristics throughout vertebrate evolution.

We have shown previously that peptide YY (PYY) receptors are uniquely distributed in various mammalian brains and also have identified the receptor from porcine hippocampal membranes as a protein of 50,000 mol wt. To extend these observations, both the characteristics of PYY-receptor interaction and the structure of the receptor have been examined and compared with those of its sister peptide, neuropeptide Y (NPY), in the brains of various vertebrates including mammals (human, dog, guinea pig, rat, and mouse), birds (chicken), reptiles (snapping turtle), amphibians (bullfrog), and fish (yellowtail fish). The affinities and relative potencies of PYY as well as NPY receptors for pancreatic polypeptide (PP) family peptides were about the same in all species examined except for chickens. PYY and NPY bound to both the PYY and NPY receptors with high affinities, but porcine and avian PPs did not. In chicken brain, however, PYY, NPY, porcine PP, and avian PP all bound to the receptors with high affinity. Analysis of the equilibrium binding data for PYY receptors produced curvilinear Scatchard plots in all of the species, suggesting the existence of high and low affinity binding sites. Affinity cross-linking using disuccinimidyl suberate followed by electrophoretic analysis of ligand-receptor complexes characterized the molecular size of PYY and NPY receptors. [125I]PYY was cross-linked to a protein of 50,000 mol wt without sulfhydryl-bonded subunits on mammalian hippocampal membranes. A receptor protein with the same mol wt was identified in other brain areas, including hypothalamus and pituitary, PYY receptors in other vertebrate brains were similar in size to those of mammalian species except in chicken brain, where a receptor protein of 67,000 mol wt was observed. In addition, we also have demonstrated that the NPY receptor is a monomeric 50,000 and 55,000 mol wt protein in mammalian and fish brains, respectively. These findings indicate that brain PYY and NPY receptors in most vertebrate species from fish to man are pharmacologically and structurally similar and have been well conserved over a period of evolution of 400 million yr. The divergence of the receptors observed in chicken brain may reflect some change in their function.     

Coupling of M2 muscarinic receptor to L-type Ca channel via c-src kinase in rabbit colonic circular smooth muscle

BACKGROUND & AIMS: The L-type Ca(2+) channel is a major pathway for Ca(2+) influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein-coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca(2+) channels via c-src kinase. METHODS: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of alpha subunit of Ca(2+) channel was determined by a GST pull-down assay. RESULTS: Methacholine (10 micromol/L) enhanced Ca(2+) channel currents by 30% under conditions whereby the M(3) receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Galphaq antibody. Similar effects were observed by blocking intracellular Ca(2+) release with heparin. Enhancement was abolished by intracellular anti-Galphai antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of alpha1c subunit of the Ca(2+) channel. In M(2) receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol. CONCLUSIONS: These studies demonstrate stimulation of Ca(2+) current in colonic smooth muscle cells by M2 receptor coupled to Galphai-G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.     

Calcium dependence of neurotensin stimulation of circular colonic muscle of the rabbit

The effect of neurotensin on smooth muscle contraction was compared in strips from rabbit proximal and distal circular colonic muscle. The effective dose for neurotensin stimulation that caused a 50% response in both tissues was similar (1.3 X 10(-10) M). The maximal isometric stress, however, was greater in the distal colon than in the proximal colon (p less than 0.01). Neurotensin stimulation of both proximal and distal colon was unaffected by tetrodotoxin, phentolamine, propranolol, naloxone, or atropine. Neurotensin-stimulated contraction was inhibited by "Ca2+-free" (pCa = 5.1) or La3+ buffer. Verapamil (10(-6) M) or nitroprusside (10(-4) M) decreased neurotensin stimulation of proximal and distal colon by approximately 40% (p less than 0.05). Removal of Ca2+ from the buffer inhibited stimulation of muscle contraction by high extracellular potassium [( K+]o) more than bethanechol stimulation (p less than 0.01). La3+ (1 mM) inhibited the contraction stimulated by bethanechol or increased [K+]o. Although verapamil inhibited contraction by bethanechol and increased [K+]o by approximately 50%, nitroprusside had no effect on the contraction mediated by these stimulants. 8-Bromo-guanosine 3',5'-cyclic monophosphate (cGMP) inhibited neurotensin, but not [K+]o or bethanechol-stimulated contraction. These data suggest (a) neurotensin stimulated colonic contractions at a concentration that is potentially physiologic, (b) neurotensin stimulated colonic smooth muscle directly without neural mediation, (c) neurotensin stimulation of colonic muscle is controlled by [Ca2+]o and [cGMP]i.     

Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest.

The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.     

钙稳态失衡在致结肠平滑肌收缩性改变中的作用

目的　探讨应激大鼠结肠平滑肌收缩时细胞内外钙离子 (Ca2 +)利用异常、细胞内钙稳态失衡在导致其收缩性改变中的作用。方法　建立寒冷 束缚应激大鼠排便异常的动物模型 ;测定离体结肠环形平滑肌收缩张力 ;差速离心制备结肠平滑肌肌浆网 ,测定肌浆网Ca2 + ATP酶活性。结果　应激大鼠结肠平滑肌收缩活性明显增强 ,并受Ca2 +通道阻滞剂显著抑制。应激大鼠结肠平滑肌肌浆网Ca2 + ATP酶活性降低 5 6 % (P <0 .0 5 )。结论　应激大鼠结肠环形平滑肌收缩活性显著增强 ,可能和肌细胞收缩时细胞外Ca2 +内流增加 ,肌浆网贮存Ca2 +释放减少、Ca2 + ATP酶活性降低等因素导致细胞内钙稳态失衡有关     

Neural contractions in colonic strips from patients with diverticular disease: role of endocannabinoids and substance P

BACKGROUND AND AIMS: Diverticulosis is a common disease of not completely defined pathogenesis. Motor abnormalities of the intestinal wall have been frequently described but very little is known about their mechanisms. We investigated in vitro the neural response of colonic longitudinal muscle strips from patients undergoing surgery for complicated diverticular disease (diverticulitis). METHODS: The neural contractile response to electrical field stimulation of longitudinal muscle strips from the colon of patients undergoing surgery for colonic cancer or diverticulitis was challenged by different receptor agonists and antagonists. RESULTS: Contractions of colonic strips from healthy controls and diverticulitis specimens were abolished by atropine. The beta adrenergic agonist (-) isoprenaline and the tachykinin NK1 receptor antagonist SR140333 had similar potency in reducing the electrical twitch response in controls and diseased tissues, while the cannabinoid receptor agonist (+)WIN 55,212-2 was 100 times more potent in inhibiting contractions in controls (IC50 42 nmol/l) than in diverticulitis strips. SR141716, a selective antagonist of the cannabinoid CB1 receptor, had no intrinsic activity in control preparations but potentiated the neural twitch in diseased tissues by up to 196% in a concentration dependent manner. SR141716 inhibited (+)WIN 55,212-2 induced relaxation in control strips but had no efficacy on (+)WIN 55,212-2 responses in strips from diverticular disease patients. Colonic levels of the endogenous ligand of cannabinoid and vanilloid TRPV1 receptors anandamide were more than twice those of control tissues (54 v 27 pmol/g tissue). The axonal conduction blocker tetrodotoxin had opposite effects in the two preparations, completely inhibiting the contractions of control strips but potentiating those in diverticular preparations, an effect selectively inhibited by SR140333. CONCLUSIONS: Neural control of colon motility is profoundly altered in patients with diverticulitis. Their raised levels of anandamide, apparent desensitisation of the presynaptic neural cannabinoid CB1 receptor, and the SR141716 induced intrinsic response, suggest that endocannabinoids may be involved in the pathophysiology of complications of colonic diverticular disease.     

Effect of pinaverium bromide on stress-induced colonic smooth muscle contractility disorder in rats

AIM: To investigate the effect of pinaverium bromide, a L-type calcium channel blocker with selectivity for the gastrointestinal tract on contractile activity of colonic circular smooth muscle in normal or cold-restraint stressed rats and its possible mechanism. METHODS: Cold-restraint stress was conducted on rats to increase fecal pellets output. Each isolated colonic circular muscle strip was suspended in a tissue chamber containing warm oxygenated Tyrode-Ringer solution. The contractile response to ACh or KCl was measured isometrically on ink-writing recorder. Incubated muscle in different concentrations of pinaverium and the effects of pinaverium were investigated on ACh or KCl-induced contraction. Colon smooth muscle cells were cultured from rats and (Ca(2+))(i) was measured in cell suspension using the Ca(2+) fluorescent dye fura-2/AM. RESULTS: During stress, rats fecal pellet output increased 61 % (P<0.01). Stimulated with ACh or KCl, the muscle contractility was higher in stress than that in control. Pinaverium inhibited the increment of (Ca(2+))(i) and the muscle contraction in response to ACh or KCl in a dose dependent manner. A significant inhibition of pinaverium to ACh or KCl induced (Ca(2+))(i) increment was observed at 10(-6) mol/L. The IC(50) values for inhibition of ACh induced contraction for the stress and control group were 1.66X10(-6) mol/L and 0.91X10(-6) mol/L, respectively. The IC(50) values for inhibition of KCl induced contraction for the stress and control group were 8.13X10(-7) mol/L and 3.80X10(-7) mol/L, respectively. CONCLUSION: Increase in (Ca(2+))(i) of smooth muscle cells is directly related to the generation of contraction force in colon. L-type Ca(2+) channels represent the main route of Ca(2+) entry. Pinaverium inhibits the calcium influx through L-type channels; decreases the contractile response to many kinds of agonists and regulates the stress-induced colon hypermotility.     

Neuropeptide Y and peptide YY, but not pancreatic polypeptide, substance P, cholecystokinin and gastric inhibitory polypeptide, inhibit the glucagon- and noradrenaline-dependent increase in glucose output in rat liver

OBJECTIVE: The gastrointestinal peptides neuropeptide Y (NPY), peptide YY (PYY), pancreatic polypeptide (PP), substance P (SP), cholecystokinin (CCK) and gastric inhibitory polypeptide (GIP) are released into the portal vein mainly during the absorptive phase. Their direct actions and their hormone modulatory effects on liver carbohydrate metabolism were investigated. METHODS: Isolated rat liver, single-pass-perfused via both the hepatic artery (120 cm H2O, 30% flow) and the portal vein (20 cm H2O, 70% flow) with a Krebs-Henseleit buffer containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate, NPY (5 nM), PYY (5 nM), PP (5 nM), SP (100 nM), CCK (100 nM) and GIP (10 nM) was infused for 10 min via either vessel. In additional experiments, insulin (100 nM), glucagon (1 nM) or noradrenaline (1 microM) were applied for 5 min via the portal vein during a 20 min portovenous infusion of one of the peptides. RESULTS: Under basal conditions, neither arterial nor portal NPY, PYY, PP, SP, CCK or GIP modified hepatic glucose and lactate metabolism. Also, none of the peptides enabled an action of portal insulin in the normally insulin-insensitive isolated perfused rat liver. NPY and PYY, but not PP, SP, CCK or GIP, inhibited the increase in glucose release by glucagon and noradrenaline. Under basal conditions, none of the peptides altered hepatic flow. Only portal NPY and PYY enhanced slightly the noradrenaline-dependent reduction of portal flow. CONCLUSIONS: NPY, PYY, PP, SP, CCK and GIP do not act directly as regulators of basal hepatic carbohydrate metabolism. NPY and PYY act as signal factors of the absorptive phase function as antagonists of the postabsorptive glucose regulatory hormones glucagon and noradrenaline.     

Effects of nociceptin/orphanin FQ on rats with cathartic colon

AIM To demonstrate the change and effect of nociceptin/orphanin FQ in the colon of rats with cathartic colon.METHODS The cathartic colon model was established by feeding rats rhubarb for 3 mo, the changes of colonic electromyography were investigated by both suspension muscle strips test and serosal recordings of colonic myoelectrical activity. Immunohistochemical staining (S-P methods) and image analysis were used to determine the changes of nociceptin/orphanin FQ in the proximal colon and distal colon of rats with cathartic colon.RESULTS Suspension muscle strips test in vitro showed OFQ (10-9-10-6 mol/L) concentration dependently caused an immediate tonic contraction in the isolated colon. But the increase of tension in cathartic colon was less than control groups (P＜0.01). Intravenous administration of OFQ (1μg/kg) caused phasic contractions in the proximal colon, while the amplitude of phasic contractions caused by OFQ in cathartic colon was much lower than that in the control groups (2.58 ± 0.41 vs 4.16± 0.53, t= -2.6, P = 0.012). OFQ was highly expressed in the myenteric plexus of the rat colon but not in the muscle cells. The immunoreactivity of OFQ in the proximal colon in cathartic colon rats decreased significantly compared with the control group (P = 0.001).CONCLUSION Colonic smooth muscle of cathartic colon showed low sensitivity to the stimulation of OFQ, suggesting that it might be caused by the abnormal distribution of OFQ or the abnormalities of receptors, leading to the disorganization of dynamic and incoordinated contractions.     

Abnormal levels of neuropeptide Y and peptide YY in the colon in irritable bowel syndrome

OBJECTIVE: To assess the levels of gut peptides involved in gastrointestinal motor, secretory and sensory function in colonic biopsies in irritable bowel syndrome (IBS) patients and healthy controls. METHODS: We studied 34 patients with IBS and 15 subjects without gastrointestinal symptoms. The predominant bowel pattern in the IBS patients was constipation in 17 patients (IBS-C) and diarrhoea in 17 patients (IBS-D). With radioimmunoassay, the levels of vasoactive intestinal peptide (VIP), substance P, neuropeptide Y (NPY) and peptide YY (PYY) were analysed in biopsies from the descending colon and ascending colon obtained during colonoscopy. RESULTS: The IBS patients had lower levels of PYY in the descending colon than the controls, but the levels in the ascending colon did not differ. The NPY levels were lower in IBS-D than in IBS-C, both in the ascending colon and in the descending colon. Low levels of VIP were more common in IBS patients, but mean levels did not differ between groups. No group differences were observed for substance P. The levels of VIP, substance P and NPY were higher in the ascending colon than in the descending colon, whereas the opposite pattern was seen for PYY. CONCLUSION: IBS patients demonstrate lower levels of PYY in the descending colon than controls. Colonic NPY levels differ between IBS subgroups based on the predominant bowel pattern. These findings may reflect the pathophysiology of IBS and the symptom variation within the IBS population.     

Sources of calcium in neurokinin A–induced contractions of human colonic smooth muscle in vitro

OBJECTIVES: Tachykinins have been implicated in the pathogenesis of colonic dysmotility. The sources of activator calcium for neurokinin A (NKA)-induced contraction of human colonic smooth muscle have not been assessed. We evaluated the contribution of extracellular and intracellular Ca2+ to NKA-induced contractions. METHODS: Circular smooth muscle strips of human colon were suspended under 1 g of tension in organ baths containing Krebs solution at 37 degrees C gased with 95% O2/5% CO2. Contractile activity was recorded isometrically. RESULTS: Cumulatively applied NKA (0.1 nmol/L-0.3 micromol/L), produced concentration-dependent contractions of human colonic smooth muscle strips that were not affected by tetrodotoxin (1 micromol/L). The contractile response to NKA was abolished in a Ca2+-free medium containing ethylenediaminetetraacetate (EDTA) (1 mmol/L). Pretreatment of muscle strips with nifedipine (1 micromol/L), an L-type voltage-operated Ca2+ channel antagonist, abolished the contractile responses to NKA. Pretreatment with SK&F 96365 (10 micromol/L and 30 micromol/L), a putative receptor-activated and voltage-operated Ca2+ channel antagonist, attenuated the contractile responses. Depletion of intracellular Ca2+ stores with thapsigargin (1 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ ATP-ase, had no effect on NKA-induced contractions. CONCLUSIONS: NKA-mediated contraction of human colonic smooth muscle is dependent on an influx of extracellular Ca2+ through L-type voltage-operated Ca2+ channels. Intracellular Ca2+ release seems to have little role to play in NKA-mediated contractions.     

Effect of areca on contraction of colonic muscle strips in rats

AIM: To investigate the effects of areca on the contractileactivity of isolated colonic muscle strips in rats andmechanism involved.METHODS: Each strip (LMPC, longitudinal muscle ofproximal colon; CMPC, circular muscle of proximal colon;LMDC, longitudinal muscle of distal colon; CMlC, circularmuscle of distal colon. ) was suspended in a tissue chambercontaining 5 mL Krebs solution (37 ℃), bubbledcontinuously with 950 mL@ L-1 O2 and 50 mL@ L-1 CO2 . Themean contractile amplitude (A), the resting tension (T),and the contractile frequency (F) were simultaneouslyrecorded on recorders.RESULTS: Arsca dose dependently increased the meancontractile amplitude, the resting tension of proximal anddistal colonic smooth muscle strips in rats ( P ＜ 0.05). Italso partly increased the contractile frequency of colonicsmooth muscle strips in rats ( P ＜ 0.05). The effects werepartly inhibited by atropine (the resting tension of LMPCdecreased from 0. 44 ± 0. 12 to 0. 17 ± 0.03; the restingtension of LMDC decreased from 0.71 ± 0.14 to 0.03 ± 0.01;the mean contractile amplitude of LMPC increased from -45.8 ± 7.2 to -30.5 ± 2.9; the motility index of CMDC decreasedfrom 86.6± 17.3 to 32.8 ± 9.3; P＜ 0.05 vs areca), but theeffects were not inhibited by hexamethonium (P＞ 0.05).CONCLUSION: Areca stimulated the motility of isolatedcolonic smooth muscle strips in rats. The stimulation ofareca might be relevant with M reoeptor partly.     

Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype.

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7-2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist [Leu31,Pro34]NPY (0.7-2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7-7 nmol) as well as the mixed Y2-Y5 agonist PYY3-36 (0.7-7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion. [D-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6-100 microg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 microg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3-36, human PP, [D-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific [125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of [D-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.     

Involvement of Src family protein tyrosine kinases in Ca(2+) sensitization of coronary artery contraction mediated by a sphingosylphosphorylcholine-Rho-kinase pathway

We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca(2+) sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca(2+)](i) levels and the Ca(2+)-dependent contraction induced by high K(+) depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase-dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca(2+) sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.