1,721,011 research outputs found
Activation and internalization of the mu-opioid receptor by the newly discovered endogenous agonists, endomorphin-1 and endomorphin-2
No full textThe multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosi
Evidence that PAR-1 and PAR-2 mediate prostanoid-dependent contraction in isolated guinea-pig gallbladder
No full text1 We have investigated the ability of protease-activated receptor-1 (PAR-1), PAR-2, PAR-3 and
PAR-4 agonists to induce contractile responses in isolated guinea-pig gallbladder. Thrombin,
trypsin, mouse PAR-1 activating (SFLLRN-NH2) peptide, and mouse PAR-2 activating (SLIGRL-
NH2) and human PAR-2 activating (SLIGKV-NH2) peptides produced a concentration-dependent
contractile response.
2 Mouse PAR-4 activating (GYPGKF-NH2) peptide, the mouse PAR-1 reverse (NRLLFS-NH2)
peptide, the mouse PAR-2 reverse (LRGILS-NH2) and human PAR-2 reverse (VKGILS-NH2)
peptides caused negligible contractile responses at the highest concentrations tested.
3 An additive e ect was observed following the contractile response induced by either trypsin or
thrombin, with the addition of a di erent PAR agonist (SFLLRN-NH2 and SLIGRL-NH2,
respectively). Desensitization to PAR-2 activating peptide attenuated the response to trypsin but
failed to attenuate the response to PAR-1 agonists, and conversely desensitization to PAR-1
attenuated the response to thrombin but failed to alter contractile responses to PAR-2 agonists.
4 The contractile responses produced by thrombin, trypsin, SFLLRN-NH2 and SLIGRL-NH2
were markedly reduced in the presence of the cyclo-oxygenase inhibitor, indomethacin, whilst the
small contractile response produced by NRLLFS-NH2 and LRGILS-NH2 were insensitive to
indomethacin.
5 The contractile responses to thrombin, trypsin, SFLLRN-NH2 and SLIGRL-NH2 were
una ected by the presence of: the non-selective muscarinic antagonist, atropine; the nitric oxide
synthase inhibitor, L-NAME; the sodium channel blocker, tetrodotoxin; the combination of selective
tachykinin NK1 and NK2 receptor antagonists, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphe-
nylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)-
N-methyl-N-[4-acetylamino-4-phenylpiperidino-2-(3,4-dichlorophenyl)-butyl] benzamide (SR48968),
respectively.
6 The results indicate that PAR-1 and PAR-2 activation causes contractile responses in the guinea-
pig gallbladder, an e ect that is mediated principally by prostanoid release, and is independent of
neural mechanisms
Deletion of Neutral Endopeptidase exacerbates intestinal inflammation induced by Clostridium difficile Toxin A
No full textExpression and function of proteinase-activated Receptor 2 in human bronchial smooth muscle
No full textTrypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction
Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways
No full textThe protease activated receptor-2 (PAR-2) belongs to a family of
G-protein–coupled receptors that are activated by proteolysis. Trypsin
cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL)
that activates the receptor. Messenger RNA (mRNA) for PAR-2 was
found in guinea pig airway tissue by reverse transcription–polymerase
chain reaction, and PAR-2 was found by immunohistochemistry
in airway epithelial and smooth-muscle cells. In anesthetized
guinea pigs, trypsin and SLIGRL-NH2 (given intratracheally or intravenously)
caused a bronchoconstriction that was inhibited by
the combination of tachykinin-NK1 and -NK2 receptor antagonists
and was potentiated by inhibition of nitric oxide synthase (NOS).
Trypsin and SLIGRL-NH2 relaxed isolated trachea and main bronchi,
and contracted intrapulmonary bronchi. Relaxation of main
bronchi was abolished or reversed to contraction by removal of
epithelium, administration of indomethacin, and NOS inhibition.
PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor
action of either trypsin or SLIGRL-NH2, because ligands of these receptors
were inactive either in vitro or in vivo, and because thrombin
(a PAR-1 and PAR-3 agonist) did not show cross-desensitization
with PAR-2 agonists in vivo. Thus, we have localized PAR-2
to the guinea-pig airways, and have shown that activation of
PAR-2 causes multiple motor effects in these airways, including
in vivo bronchoconstriction, which is in part mediated by a neural
mechanism
Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers
No full text
Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome.
No full textBACKGROUND & AIMS: Intestinal mast cell infiltration may participate to abdominal pain in irritable bowel syndrome (IBS) patients. However, the underlying mechanisms remain unknown. We assessed the effect of mast cell mediators released from the colonic mucosa of IBS patients on the activation of rat sensory neurons in vitro.
METHODS: Colonic mast cell infiltration and mediator release were assessed with quantitative immunofluorescence and immunoenzymatic assays. The effect of mucosal mediators was tested on mesenteric sensory nerve firing and Ca(2+) mobilization in dorsal root ganglia in rats.
RESULTS: Mediators from IBS patients, but not controls, markedly enhanced the firing of mesenteric nerves (14.7 +/- 3.2 imp/sec vs 2.8 +/- 1.5 imp/sec; P < .05) and stimulated mobilization of Ca(2+) in dorsal root ganglia neurons (29% +/- 4% vs 11% +/- 4%; P < .05). On average, 64% of dorsal root ganglia responsive to mediators were capsaicin-sensitive, known to mediate nociception. Histamine and tryptase were mainly localized to mucosal mast cells. IBS-dependent nerve firing and Ca(2+) mobilization were correlated with the area of the colonic lamina propria occupied by mast cells (r = 0.74; P < .01, and r = 0.78; P < .01, respectively). IBS-dependent excitation of dorsal root ganglia was inhibited by histamine H(1) receptor blockade and serine protease inactivation (inhibition of 51.7%; P < .05 and 74.5%; P < .05; respectively).
CONCLUSIONS: Mucosal mast cell mediators from IBS patients excite rat nociceptive visceral sensory nerves. These results provide new insights into the mechanism underlying visceral hypersensitivity in IBS
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