Predominance of delta-opioid-binding sites in the porcine enteric nervous system. (25/208)

The antidiarrheal and constipating actions of opioids are mediated in part by enteric neurons, which lie within the wall of the small intestine and colon, but the differential expression of specific, high-affinity opioid-binding sites in ganglionated plexuses within functionally distinct intestinal segments has not been examined. We determined the saturation binding characteristics under Na+-free conditions of the nonselective opioid receptor (OPR) ligand [3H][(5alpha,7alpha)-17-(cyclopropylmethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy-6-m ethoxy-alpha, alpha-dimethyl-6,14-ethenomorphinan-7-methanol] (diprenorphine) and the respective delta-, kappa-, and mu-OPR ligands [3H]naltrindole, D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8 -yl]benzeneacetamide ([3H]U-69,593), and [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) in neuronal membranes isolated from myenteric and submucosal plexuses of porcine small intestine and colon. Naloxone-displaceable [3H]diprenorphine-binding sites (KD values ranging from 0.2-0.5 nM and Bmax = 50-95 fmol/mg of protein) were found in both subregions from all gut segments examined. High-affinity [3H]naltrindole sites (KD = 60-140 pmol) were at highest densities (approximately 60 fmol/mg of protein) in submucosal plexus of the ileum and distal colon myenteric plexus and were at lowest densities (8-9 fmol/mg of protein) in the submucosal plexuses of cecum and distal colon. [3H]U-69,593 sites (KD = 0.3-4 nM) were present only in the myenteric plexuses of all segments examined, with highest densities in cecum and proximal colon (44-47 fmol/mg of protein). [3H]DAMGO-binding sites were expressed at relatively low densities in the enteric plexuses of all gut regions. These results indicate that delta-OPRs predominate in the porcine enteric nervous system with a more circumscribed expression of kappa- and mu-OPRs.  (+info)

Muscarinic receptor antagonist-induced lenticular opacity in rats. (26/208)

Investigations on compound A, an M2-sparing M3 muscarinic receptor antagonist, showed that focal polar anterior subcapsular lenticular opacities, characterized by focal epithelial proliferation, developed in Sprague-Dawley rats. The incidence and bilateral localization of this change increased generally with dose and time, though plateauing after 8 months of treatment; however the severity progressed very slightly. Over a 1-year period, no anterior cortical lens fiber changes or other histological ocular changes developed. A decreased severity of the change and apoptosis suggested some regression after a 26-week recovery period. Two nonselective muscarinic receptor antagonists, atropine and tolterodine, induced similar lenticular changes in rats. A hypothesis in relation to an indirect effect of the drug, such as increased illumination of the lens due to mydriasis observed with all these compounds, was investigated and disproven. Because these opacities are induced by structurally unrelated muscarinic receptor antagonists (atropine and tolterodine), it is likely that these lenticular changes are the result of muscarinic receptor inhibition. However, hypotheses regarding a direct effect of the drug on muscarinic receptors in the lens epithelium, possibly mediated by drug and/or metabolite(s) in the aqueous humor and/or lens epithelium, remain to be investigated. This lenticular opacity is similar to that observed spontaneously in Sprague-Dawley rats, although the latter occur at a lower incidence. No such lenticular opacities have been reported in other animal species, including man, after treatment with muscarinic receptor antagonists.  (+info)

Characterization of mu, kappa, and delta opioid binding in amphibian whole brain tissue homogenates. (27/208)

Opioid agonists produce analgesia in mammals through the activation of mu, kappa, or delta opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that mu, kappa, or delta opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin) (mu-selective), [(3)H]U65953 [(5 alpha, 7 alpha,8 beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamid e] (kappa-selective), and [(3)H]DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (delta-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (K(D)) in the low nanomolar range. The density of opioid binding sites (B(max)) was 224.4, 118.6, and 268.9 fmol/mg for mu, kappa, and delta opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the kappa radioligand bound to significantly fewer sites than did the mu or delta radioligands. K(i) values for unlabeled mu, kappa, and delta competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile. The present data suggest that mu, kappa, and delta opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.  (+info)

In vitro microbiological evaluation of TEI-1194 and TEI-2012, novel antipseudomonal semisynthetic penicillins. (28/208)

TEI-1194, sodium 6-[D-(-)-alpha-(coumarin-3-carboxamide)-phenylacetamide] penicillanate and TEI-2012, sodium 6[D-(-)alpha-(8-hydroxy-coumarin-3-carboxamide)-phenylacetamide] penicillanate are new semisynthetic penicillin derivatives both possessing a broad spectrum of in vitro antibacterial activities. Minimal inhibitory concentrations of both agents were compared with carbenicillin. TEI-1194 and TEI-2012 were clearly found to have more potent activities especially against Pseudomonas aeruginosa than carbenicillin. At a concentration at 6.25 micrograms/ml, 85 approximately 90% of a total of 50 strains of clinically isolated P. aeruginosa were inhibited by TEI-1194 and TEI-2012, whereas carbenicillin had no effect. Evaluation of the antibacterial activity against a series of mutants producing different levels of beta-lactamases and test of the susceptibilities to some beta-lactamases demonstrated that TEI-1194 and TEI-2012 had low susceptibility to various cephalosporinases. However, both compounds were susceptible to penicillinase from Klebsiella pneumoniae H-2 at a rate of about 15% of penicillin-G taking its absolute rate as 100.  (+info)

Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50. (29/208)

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.  (+info)

Topical nepafenac inhibits ocular neovascularization. (30/208)

PURPOSE: Topical nepafenac readily penetrates the cornea and is metabolized to amfenac, a potent cyclooxygenase (COX)-1 and COX-2 inhibitor. In this study, we tested the effect of topical nepafenac in three murine models of ocular neovascularization (NV). METHODS: A masked trial was performed to compare the topical effects of vehicle with one of several concentrations of nepafenac (0.01%, 0.03%, 0.1%, or 0.5%), 0.1% diclofenac, or 0.5% ketorolac tromethamine in mice with oxygen-induced ischemic retinopathy, mice with choroidal NV (CNV) due to laser-induced rupture of Bruch's membrane, or transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF transgenic mice). RESULTS: Mice treated with 0.1% or 0.5% nepafenac had significantly less CNV and significant less ischemia-induced retinal NV than did vehicle-treated mice. Nepafenac also blunted the increase in VEGF mRNA in the retina induced by ischemia. In rho/VEGF transgenic mice, nepafenac failed to inhibit neovascularization. In additional studies, compared with vehicle-treated mice, mice treated with 0.1% or 0.03% nepafenac had significantly less CNV, whereas eyes treated with 0.1% diclofenac showed no significant difference. Mice treated with 0.5% ketorolac tromethamine for 14 days had high mortality, but when evaluated after 7 days of treatment showed no difference from mice treated with vehicle for 7 days. CONCLUSIONS: Topical nepafenac inhibits CNV and ischemia-induced retinal neovascularization by decreasing production of VEGF. The absence of effect in rho/VEGF transgenic mice is consistent with this mechanism. Topical nepafenac may provide an effective new treatment for ocular neovascularization. The excellent corneal penetration of nepafenac certainly plays an important role in this effect. It is possible that other antiangiogenic agents are also amenable to topical application after formulations are identified that maximize their corneal penetration. Because of the many advantages of the topical route of delivery, this is a possible topic for exploration.  (+info)

Kappa-opioid receptor-mediated enhancement of the hyperpolarization-activated current (I(h)) through mobilization of intracellular calcium in rat nucleus raphe magnus. (31/208)

The hyperpolarization-activated current (Ih) is important in the control of resting membrane potential, in the regulation of network firing pattern and in the modulation of presynaptic transmitter release in central neurons. Recent studies on native and cloned Ih channels have demonstrated that the Ih channel is commonly modulated by cAMP through a positive shift in its voltage dependence without a change in its maximum current. The present study demonstrates that activation of kappa-opioid receptors enhances Ih by increasing its maximum current in brainstem neurons in the nucleus raphe magnus. Agents that interfere with the release of intracellular calcium from calcium stores altered the maximum Ih and significantly attenuated the kappa-receptor-mediated enhancement of Ih. These results suggest that kappa-opioid receptors enhance the maximum Ih by mobilizing intracellular calcium from calcium stores. This provides a physiological function for kappa-receptor-stimulated calcium release and may suggest another Ih-regulating mechanism by intracellular calcium in central neurons.  (+info)

Phosphorylation- and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by nonredox-type inhibitors. (32/208)

Nonredox-type 5-lipoxygenase (5-LO) inhibitors such as ZM230487 or L-739.010 potently suppress leukotriene biosynthesis at low cellular peroxide tone. Here, we show that inhibition of 5-LO product formation by nonredox-type 5-LO inhibitors in human isolated polymorphonuclear leukocytes (PMNL) depends on the activation pathway of 5-LO. Thus, compared with 5-LO product synthesis induced by the Ca2+-mobilizing agent ionophore A23187, cell stress-induced 5-LO product formation involving 5-LO kinase pathways required ~10- to 100-fold higher concentrations of ZM230487 or L-739.010 for comparable 5-LO inhibition. No such differences were observed for the iron ligand-type 5-LO inhibitor BWA4C or the novel-type 5-LO inhibitors hyperforin and 3-O-acetyl-11-keto-boswellic acid. Experiments using purified 5-LO revealed that Ca2+ is no prerequisite for potent enzyme inhibition by ZM230487, and exposure of PMNL to the combination of ionophore and cell stress did not restore potent 5-LO suppression. Intriguingly, a significant difference in the potency of nonredox-type inhibitors (but not of BWA4C) was determined between wild-type 5-LO and the mutant S271A/S663A-5-LO (lacking phosphorylation sites for ERK1/2 and MAPKAPK-2) in HeLa cells. Collectively, our data suggest that compared with Ca2+-mediated 5-LO product formation, enzyme activation involving 5-LO phosphorylation events specifically and strongly alters the susceptibility of 5-LO toward nonredox-type inhibitors in intact cells.  (+info)