Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding. (1/262)

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.  (+info)

A-Current down-modulated by sigma receptor in frog pituitary melanotrope cells through a G protein-dependent pathway. (2/262)

Gramicidin perforated patch-clamp recordings were used to study the effects of two sigma 1 receptor ligands, (+)-N-cyclopropylmethyl-N-methyl-1, 4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO 1784) and (+)-pentazocine, on the transient outward potassium current (IA) in cultured frog melanotrope cells. (+)-Pentazocine reversibly decreased the current amplitude in a dose-dependent manner. The effects of (+)-pentazocine were mimicked by JO 1784 and were markedly reduced by the sigma 1 receptor antagonist, N, N-dipropyl-2-[4-methoxy-3-2(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE 100). Inactivation rate of IA was best fitted with a double exponential function, yielding time constants of 23.7 and 112.5 ms. (+)-Pentazocine (20 microM) accelerated the current decay, decreasing the time constants to 10.7 and 59 ms, respectively. Current-voltage experiments revealed that (+)-pentazocine (20 microM) did neither modify the open-state I/V curves nor the voltage dependence of IA. However, (+)-pentazocine (20 microM) shifted the steady-state inactivation curve toward more negative potentials and increased the time constant of the time-dependent removal of inactivation. In whole-cell experiments, internal dialysis of guanosine-5'-O-(3-thiophosphate) (100 microM) irreversibly prolonged the response to (+)-pentazocine. In addition, cholera toxin pretreatment (1 microgram. ml-1; 12 h) suppressed the inhibition of IA by (+)-pentazocine (20 microM). It is concluded that in frog melanotrope cells, a cholera toxin-sensitive, G protein-dependent inhibition of IA through a sigma 1 receptor activation, at least partially, underlies the excitatory effect of sigma ligands.  (+info)

Sigma1 recognition sites in rabbit iris-ciliary body: topical sigma1-site agonists lower intraocular pressure. (3/262)

In this study, we examined the presence of sigma1 and sigma2 sites in the rabbit iris-ciliary body by receptor binding and investigated their effects on intraocular pressure (IOP) in albino rabbits. The iris-ciliary body has binding sites for the sigma1-site agonist [3H](+)-pentazocine (Kd = 4.6 nM; Bmax = 212 fmol/mg protein) and sigma2 sites labeled with [3H]1,3-di-o-tolylguanidine (DTG) (Kd = 8. 2 nM; Bmax = 1120 fmol/mg protein). In competition binding studies, (+)-pentazocine and the sigma antagonist NE-100 displayed high affinity for sigma1 sites (Ki = 2.1 and 2.4 nM, respectively), whereas (+)-N-allylnormetazocine (NANM) was less potent (Ki = 178 nM). Unilateral topical (+)-pentazocine (0.01-0.1%) caused a significant dose-related reduction of IOP in ocular normotensive rabbits and in the alpha-chymotrypsin model of ocular hypertension. (+)-NANM was less potent than (+)-pentazocine. Neither compound altered the IOP of the contralateral eye, and their hypotensive activity was blocked by NE-100 that, by itself, had no effect on IOP. (-)-Pentazocine, (-)-NANM, and DTG had no effect on IOP. DTG prevented the hypotensive effect of (+)-pentazocine, suggesting that it acts as a sigma1-site antagonist. sigma-Site ligands did not affect pupil diameter or cause ocular inflammation. Topical [3H](+)-pentazocine reaches the intraocular tissues within 30 min, and its uptake in the iris-ciliary body and retina was significantly reduced by topical pretreatment with NE-100, as expected for a receptor-specific agent. Reverse-phase HPLC confirmed the presence of intact (+)-pentazocine in iris-ciliary body homogenates. sigma1-Site agonists may offer a novel class of agents potentially effective in the control of ocular hypertension.  (+info)

Mechanism-based inactivation of rat liver cytochrome P-450 2B1 by 2-methoxy-5-nitrobenzyl bromide. (4/262)

Mechanism-based inactivators serve as probes of enzyme mechanism, function, and structure. Koshland's Reagent II (2-methoxy-5-nitrobenzyl bromide, KR-II) is a potential mechanism-based inactivator of enzymes that perform O-dealkylations. The major phenobarbital-inducible form of cytochrome P-450 in male rat liver microsomes, CYP2B1, is capable of catalyzing O-dealkylations. The interactions of KR-II with purified CYP2B1 in the reconstituted system containing P-450, NADPH:P-450 oxidoreductase, and sonicated dilaurylphosphatidyl choline were studied. The benzphetamine N-demethylase activity of CYP2B1 was inactivated by KR-II in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The inactivation also required KR-II, and the rate of activity loss was dependent on the concentration of KR-II in a saturable fashion. The inactivator concentration required for the half-maximal rate of inactivation (KI) was approximately 0.1 mM. The inactivation was not prevented by the addition of the nucleophiles dithiothreitol and glutathione, nor was it reversed by gel filtration. The present results demonstrate that KR-II is a mechanism-based inactivator of rat CYP2B1.  (+info)

Odor response properties of rat olfactory receptor neurons. (5/262)

Molecular biology studies of olfaction have identified a multigene family of molecular receptors that are likely to be involved in odor transduction mechanisms. However, because previous functional data on peripheral coding were mainly collected from inferior vertebrates, it has been difficult to document the degree of specificity of odor interaction mechanisms. As a matter of fact, studies of the functional expression of olfactory receptors have not demonstrated the low or high specificity of olfactory receptors. In this study, the selectivity of olfactory receptor neurons was investigated in the rat at the cellular level under physiological conditions by unitary extracellular recordings. Individual olfactory receptor neurons were broadly responsive to qualitatively distinct odor compounds. We conclude that peripheral coding is based on activated arrays of olfactory receptor cells with overlapping tuning profiles.  (+info)

New studies on trans-anethole oxide and trans-asarone oxide. (6/262)

The widespread use of naturally occurring alkenylbenzenes as flavoring and fragrance agents has led to a long-standing interest in their toxicity and carcinogenicity. Among them several allyl- and propenylbenzenes have been found to be mutagenic and carcinogenic. It has been shown that the carcinogenicity of several allylbenzenes can be related to the formation of electrophilic sulfuric acid esters following 1'-hydroxylation. Unlike the allylbenzenes, the mechanisms of carcinogenesis of propenylbenzenes such as anethole and asarone are not clear. It has been reported that one of the main metabolic pathways of trans-anethole is the epoxidation of the side chain 1,2-double bond, which was responsible for cytotoxicity but not for genotoxicity. However, we report here that synthetic trans-anethole oxide prepared from trans-anethole and dimethyldioxirane is not only mutagenic for Salmonella tester strains but is also carcinogenic in the induction of hepatomas in B6C3F1 mice and skin papillomas in CD-1 mice. Synthetic trans-asarone oxide was also carcinogenic in the induction of hepatomas as well as mutagenic for Salmonella strains. Further studies are needed on these side chain oxides of trans-anethole and trans-asarone as possible metabolites in the toxicity, mutagenicity and carcinogenicity of these and other propenylbenzenes.  (+info)

Dose-response trend tests for tumorigenesis, adjusted for body weight. (7/262)

Several studies have demonstrated a relationship between rodent body weight and tumor incidence for some tissue/organ sites. It is not uncommon for a chemical tested for carcinogenicity to also affect body weight. In such cases, comparisons of tumor incidence may be biased by body-weight differences across dose groups. A simple procedure was investigated for reducing this bias. This procedure divides the animals into a few groups based on body weight. Body weight at 12 months was used, before the appearance of a tumor was likely to affect body weight. Statistics for dose-response trend tests are calculated within body weight strata and pooled to obtain an overall dose-response trend test. This procedure is analogous to that currently used, of stratifying animals, based on their age at the time of removal from a study. Age stratification is used to account for differences in animal age across dose groups, which can affect comparisons of tumor incidence. Several examples were investigated where the high-dose group had reduced body weights and associated reductions in tumor incidence. When the data were analyzed by body-weight strata, some positive dose-response trends for tumor incidence were demonstrated. In one case, the weight-adjusted analysis indicated that a negative dose-response trend in tumor incidence was a real effect, in addition to a body weight reduction. These examples indicate that it is important to consider the effects of body weight changes as low as 10%, and perhaps below, that were caused by chemicals in 2-year bioassays for carcinogenesis. The simple procedure of analyzing tumor incidence within body-weight strata can reduce the bias introduced by weight differences across dose groups.  (+info)

Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest. (8/262)

We have found that the bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) induced a dose- and time-dependent apoptotic response in human leukemic cells. MTC and colchicine rapidly disrupted the microtubule integrity and arrested cells at the G2-M phase before the onset of apoptosis. These responses were mediated by microtubule inhibition because 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propionyl]amino]-2,4,6-cycloh eptatrien-1-one and lumicolchicine, inactive analogues of MTC and colchicine, respectively, were unable to promote microtubule disassembly, cell cycle arrest, and apoptosis. Although 1 microM MTC induced a complete microtubule disruption after 1 h of incubation in human leukemic HL-60 cells that led to an accumulation of cells at the G2-M phase, MTC-induced apoptosis occurred after 9 h of treatment. This indicates the existence of a rather long lag between microtubule disruption and the onset of apoptosis. Unlike colchicine, the removal of MTC during this lag resulted in rapid microtubule repolymerization, followed by restoration of normal cell cycle and cell growth. MTC, but not 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino]-2,4,6-cyclo heptatrien-1-one, induced c-jun expression as well as c-Jun NH2-terminal kinase and caspase activation, indicating that these signaling pathways are triggered by the specific action of MTC on microtubules. Caspase inhibition prevented MTC-induced apoptosis. Overexpression of bcl-2 or bcl-xL by gene transfer in human erythroleukemic HEL cells abrogated MTC-induced apoptosis, but cells remained arrested in G2-M, suggesting that bcl-2 and bcl-xL block the signaling pathway between G2-M arrest and triggering of apoptosis. MTC-treated bcl-2 and bcl-xL-transfected HEL cells recovered their capacity to proliferate after MTC removal. These results indicate that microtubule inhibition induces G2-M arrest and apoptosis in leukemic cells, showing a lag phase between G2-M arrest and the onset of apoptosis, regulated by bcl-2 and bcl-xL, during which MTC displays a reversible action on microtubule depolymerization and G2-M cell cycle arrest. Thus, MTC is a potent apoptotic inducer on human leukemic cells and shows a remarkable reversible action on microtubule network and cell cycle before commitment for apoptosis is reached.  (+info)