Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding.
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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)
Molecular and ligand-binding characterization of the sigma-receptor in the Jurkat human T lymphocyte cell line.
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The sigma binding site present in the Jurkat human T lymphocyte cell line was investigated. Jurkat cell membranes were found to have a single saturable binding site for [3H]haloperidol, a sigma ligand (dissociation constant, 3.9 +/- 0.3 nM). The binding of [3H]haloperidol was inhibited by several sigma ligands. Northern analysis and reverse transcription-polymerase chain reaction provided evidence for the expression of the recently cloned type 1 sigma-receptor (sigma-R1) in Jurkat cells. The sigma-R1 cDNA cloned from these cells was functional in heterologous expression systems. When expressed in mammalian cells, the cDNA-induced binding was saturable with dissociation constants of 1.9 +/- 0.3 nM for [3H]haloperidol and 12 +/- 2 nM for (+)-pentazocine. The binding of [3H]progesterone, a putative endogenous ligand to sigma-R1, to the Jurkat cell sigma-receptor could be directly demonstrated by using heterologously expressed sigma-R1 cDNA. The binding of [3H]progesterone was saturable, with a dissociation constant of 88 +/- 7 nM. Progesterone and haloperidol interacted with the receptor competitively. Reverse transcription-polymerase chain reaction also produced evidence for the existence of an alternatively spliced sigma-R1 variant in Jurkat cells. This splice variant was found to be nonfunctional in ligand binding assays. This constitutes the first report on the molecular characterization of the sigma-receptor in immune cells. (+info)
Modulation of amphetamine-stimulated [3H]dopamine release from rat pheochromocytoma (PC12) cells by sigma type 2 receptors.
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An important regulatory mechanism of synaptic dopamine (DA) levels is activation of the dopamine transporter (DAT), which is a target for many drugs of abuse, including amphetamine (AMPH). sigma receptors are located in dopaminergic brain areas critical to reinforcement. We found previously that agonists at sigma2 receptors enhanced the AMPH-stimulated release of [3H]DA from slices of rat caudate-putamen. In the present study, we modeled this response in undifferentiated pheochromocytoma-12 (PC12) cells, which contain both the DAT and sigma2 receptors but not neural networks that can complicate investigation of individual neuronal mechanisms. We found that enhancement of AMPH-stimulated [3H]DA release by the sigma agonist (+)-pentazocine was blocked by sigma2 receptor antagonists. Additionally, the reduction in the effect of (+)-pentazocine by the inclusion of ethylene glycol bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid led us to hypothesize that sigma2 receptor activation initiated a Ca2+-dependent process that resulted in enhancing the outward flow of DA via the DAT. The source of Ca2+ required for the enhancement of reverse transport did not appear to be via N- or L-type voltage-dependent Ca2+ channels, because it was not affected by nitrendipine or omega-conotoxin. However, two inhibitors of Ca2+/calmodulin-dependent protein kinase II blocked enhancement in AMPH-stimulated release by (+)-pentazocine. Our findings suggest that sigma2 receptors are coupled to the DAT via a Ca2+/calmodulin-dependent protein kinase II transduction system in PC12 cells, and that sigma2 receptor antagonists might be useful in the treatment of drug abuse by blocking elevation of DA levels via reversal of the DAT. (+info)
A-Current down-modulated by sigma receptor in frog pituitary melanotrope cells through a G protein-dependent pathway.
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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)
Assessment of opioid partial agonist activity with a three-choice hydromorphone dose-discrimination procedure.
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The discriminative stimulus and subjective effects of opioid mixed agonist-antagonists were assessed in volunteer nondependent heroin users trained in a three-choice drug discrimination procedure to discriminate among the effects of i.m. administration of 2 ml of saline, 1 mg of hydromorphone, and 4 mg of hydromorphone (a morphine-like mu agonist). Other subjective, behavioral, and physiological measures were concurrently collected. The discrimination was readily learned by six of the eight subjects. After training, generalization curves were determined for the following i.m. drug conditions: hydromorphone (0.375-4.0 mg), pentazocine (7.5-60 mg), butorphanol (0.75-6 mg), nalbuphine (3-24 mg), and buprenorphine (0.075-0.6 mg). All five of the test drugs were discriminated significantly or showed trends toward being discriminated as hydromorphone 1 mg-like at one or more dose levels. Hydromorphone showed an inverted U-shaped dose-effect function on the hydromorphone 1 mg-like discrimination. Subjective effect measures produced clearer differentiation among the test drugs than did drug discrimination performance. The present results differ from those of a previous study that observed a close relationship between the results of the discrimination measure and subjective effect measures. The previous study used similar methods and test drugs but different training drugs (e.g., 3 mg of hydromorphone versus 6 mg of butorphanol versus saline). It appears that both the sensitivity of drug discrimination performance to between-drug differences and the relationship between discriminative and subjective effects depends upon the specific discrimination that is trained (e.g., two-choice or three-choice). The present high dose-low dose-saline discrimination procedure appears useful for assessing partial agonist activity. The present data are consistent with partial agonist activity for pentazocine, butorphanol, nalbuphine, and buprenorphine. (+info)
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)
Intracellular sigma1 receptor modulates phospholipase C and protein kinase C activities in the brainstem.
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Most physiological effects of sigma1 receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the sigma1 receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the sigma1 receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C beta1 and beta2 isoforms and the phosphorylation of a protein of the same molecular weight as the cloned sigma1 receptor lead to a desensitization of the sigma1 motor response. Our results indicate that the intracellular sigma1 receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses. (+info)
The sigma-ligand (+)-pentazocine depresses M current and enhances calcium conductances in frog melanotrophs.
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Gramicidin-perforated patch clamp experiments and microfluorimetric measurements were performed to study the ionic mechanisms involved in the sigma-receptor-mediated stimulation of frog (Rana ridibunda) pituitary melanotrophs. The sigma-ligand (+)-pentazocine (50 microM) depressed a sustained outward K(+) current. The kinetic properties of this K(+) component, investigated by analyzing tail currents, were reminiscent of those of the M current (I(M)), with an activation threshold close to -60 mV, a -21-mV half-maximal activation potential, and two-component exponential deactivation kinetics at -90 mV. (+)-Pentazocine (20 microM) produced a 12-mV rightward shift of the activation curve and accelerated the deactivation rate of the tail current. It is also demonstrated that (+)-pentazocine (20 microM) reversibly increased both voltage-dependent calcium conductances and internal calcium level. Altogether, these results suggest that the sigma-receptor-induced modulation of I(M) and calcium currents likely underlies the increase of intracellular [Ca(2+)]. (+info)