The interaction of raloxifene and the active metabolite of the antiestrogen EM-800 (SC 5705) with the human estrogen receptor.
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A naturally occurring mutation at amino acid 351 (D351Y) in the human estrogen receptor (ER) can change the pharmacology of antiestrogens. Raloxifene is converted from an antiestrogen to an estrogen, whereas the biological properties of the steroidal pure antiestrogen ICI 182,780 are not affected by the D351Y ER (Levenson, A. S., and Jordan, V. C. Cancer Res., 58: 1872-1875, 1998). We propose an assay system that can be used to classify antiestrogens by determining their ability to up-regulate transforming growth factor alpha (TGF-alpha) mRNA in MDA-MB-231 cells stably transfected with either wild-type or D351Y ER. The novel compound EM-800 and its active metabolite, EM-652, have been reported to be p.o. active nonsteroidal pure antiestrogens. Using the D351Y cell line, EM-652 is able to up-regulate TGF-alpha mRNA in a dose-dependent manner and to a similar extent as estradiol, whereas in the wild-type cell line, it acts as an antiestrogen. In addition, the pure antiestrogen ICI 182,780 is capable of inhibiting EM-652-induced TGF-alpha mRNA expression at the D351Y ER. In MCF-7 cells expressing wild-type ER, it has previously been shown that ICI 182,780 decreases ER only at the protein level. EM-652 treatment does not decrease ER protein levels to a similar extent as ICI 182,780 treatment, and, in addition, EM-652 has no effect on ER mRNA levels. In proliferation assays, EM-652 is as effective as raloxifene in inhibiting cell growth. From these studies, we conclude that the reason the pharmacology of EM-652 is similar to that of raloxifene is because they both fit the ER in the same manner, and their biology depends on an interaction of the antiestrogenic side chain with amino acid 351. (+info)
A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.
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Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades. (+info)
Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6, and GluR7.
(35/1899)
In the CNS kainate subtype glutamate receptors (GluRs) are likely to be heteromeric assemblies containing multiple gene products. However, although recombinant kainate receptors from the GluR5-GluR7 gene family have been studied extensively in their homomeric forms, there have been no tests to determine whether these subunits can coassemble with each other. We used the GluR5 selective agonists (RS)-2-amino-3-(3-hydroxy-5-tertbutylisoxazol-4-yl)propanoic acid (ATPA) and (S)-5-iodowillardiine (I-will) to test for the coassembly of GluR5 with GluR6 and GluR7 by measuring changes in rectification that occur for heteromeric receptors containing both edited and unedited Q/R site subunits. Birectifying ATPA and I-will responses resulting from polyamine block for homomeric GluR5(Q) became outwardly rectifying when GluR6(R) was coexpressed with GluR5(Q), although GluR6 was not activated by ATPA or I-will, indicating the formation of heteromeric receptors. Similar approaches showed the coassembly of GluR7 with GluR6 and GluR5. Heteromeric kainate receptors containing both GluR5 and GluR6 subunits exhibited novel functional properties, including reduced desensitization and faster recovery from desensitization than those recorded for homomeric GluR5. Coexpression of GluR6 with GluR5 also enhanced the magnitude of responses to GluR5 selective agonists. In contrast, the coassembly of GluR7 with GluR6 markedly decreased the amplitude of agonist responses. Our results indicate that, similar to AMPA receptors, the kainate receptor subunits GluR5-GluR7 exhibit promiscuous coassembly. The formation of heteromeric kainate receptors may help to explain why the functional properties of native kainate receptors differ from those that have been reported for recombinant kainate receptors. (+info)
Protective effect of FR168888, a new Na+/H+ exchange inhibitor, on ischemia and reperfusion-induced arrhythmia and myocardial infarction in rats: in comparison with other cardioprotective compounds.
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We have studied the effects of FR168888 (5-hydroxymethyl-3-(pyrrol-1-yl)benzoylguanidine methanesulfonate), a new Na+/H+ exchange inhibitor, on ischemia and reperfusion-induced arrhythmia and myocardial infarction in anesthetized rats and compared them with those of other cardioprotective compounds. FR168888 had a potent inhibitory effect on Na+/H+ exchange of rat lymphocytes acidified with Na+-propionate with a Ki value of 6.4 nM. Pretreatment with FR168888 (0.032-0.32 mg/kg, i.v.) reduced or completely abolished the ventricular fibrillation (VF) induced by reperfusion after 5 min of regional ischemia, while lidocaine, a class I antiarrhythmic agent, showed less effect against VF as compared with FR168888. The size of myocardial infarction induced by 60-min ischemia and 60-min reperfusion was attenuated by FR168888 dose-dependently (1.0-10 mg/kg, i.v.), and ventricular tachycardia and VF were significantly reduced during the ischemic period. In contrast, propranolol and diltiazem did not show such protective effects on myocardial infarct size. In addition, FR168888 did not change hemodynamic parameters in rats. These results indicate that FR168888 has a strong inhibitory effect on Na+/H+ exchange and that treatment with FR168888 can protect the heart from arrhythmia and myocardial cell death in ischemic and reperfused situations. (+info)
Combined mechanical trauma and metabolic impairment in vitro induces NMDA receptor-dependent neuronal cell death and caspase-3-dependent apoptosis.
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Neuronal necrosis and apoptosis occur after traumatic brain injury (TBI) in animals and contribute to subsequent neurological deficits. In contrast, relatively little apoptosis is found after mechanical injury in vitro. Because in vivo trauma models and clinical head injury have associated cerebral ischemia and/or metabolic impairment, we transiently impaired cellular metabolism after mechanical trauma of neuronal-glial cultures by combining 3-nitropropionic acid treatment with concurrent glucose deprivation. This produced greater neuronal cell death than mechanical trauma alone. Such injury was attenuated by the NMDA receptor antagonist dizocilpine (MK801). In addition, this injury significantly increased the number of apoptotic cells over that accruing from mechanical injury alone. This apoptotic cell death was accompanied by DNA fragmentation, attenuated by cycloheximide, and associated with an increase in caspase-3-like but not caspase-1-like activity. Cell death was reduced by the pan-caspase inhibitor BAF or the caspase-3 selective inhibitor z-DEVD-fmk, whereas the caspase-1 selective inhibitor z-YVAD-fmk had no effect; z-DEVD-fmk also reduced the number of apoptotic cells after combined injury. Moreover, cotreatment with MK801 and BAF resulted in greater neuroprotection than either drug alone. Thus, in vitro trauma with concurrent metabolic inhibition parallels in vivo TBI, showing both NMDA-sensitive necrosis and caspase-3-dependent apoptosis. (+info)
High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.
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Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h. (+info)
Kinetics of endothelin-1 binding in the dog liver microcirculation in vivo.
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Endothelin-1 (ET-1) is a 21-amino acid peptide produced by vascular endothelial cells that acts as a potent constrictor of hepatic sinusoids. Hepatic binding of tracer (125)I-labeled ET-1 was investigated in anesthetized dogs with the multiple-indicator dilution technique with simultaneous measurements of unlabeled immunoreactive ET-1 plasma levels. Despite 80% binding to albumin, tracer (125)I-ET-1 was avidly extracted by the liver, with only 15 +/- 6% of the peptide surviving passage through the organ. Exchange of ET-1 between plasma and binding sites, probably located on the surface of liver cells, was quantitatively described by a barrier-limited, space-distributed variable transit time model. Reversible and irreversible parallel binding sites were found. Reversible and irreversible plasma clearances of unbound (125)I-ET-1 were 0.084 +/- 0.033 ml. s(-1). g liver(-1) and 0.17 +/- 0.09 ml. s(-1). g liver(-1), respectively, and the dissociation rate constant for reversible binding was 0.24 +/- 0.12 s(-1). The specific ET(A) receptor antagonist BMS-182874 did not modify binding to either site. The nonspecific ET(A)/ET(B) antagonist LU-224332 dose-dependently reduced irreversible binding only. ET-1 levels in the hepatic vein were significantly lower than in the portal vein but were not different from those in the hepatic artery. The ratio between hepatic vein and portal vein levels (0.64 +/- 0.31) was considerably higher than survival fractions, suggesting a substantial simultaneous release of newly synthesized or stored ET-1 by the liver. These results demonstrate both substantial clearance and production of ET-1 by the intact liver. Hepatic ET-1 clearance is mediated by the ET(B) receptor, with the presence of reversible, nonspecific ET-1 binding at the liver surface (+info)
Interactive effects of central leptin and peripheral fuel oxidation on estrous cyclicity.
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A 48-h period of fasting inhibits estrous cycles in Syrian hamsters, and fasting-induced anestrus can be prevented by intracerebroventricular treatment with leptin during the fasting period. In the present experiment, the effects of intracerebroventricular leptin were blocked by systemic treatment with inhibitors of metabolic fuel oxidation. Leptin was infused continuously into the lateral ventricles (1 microgram/day) during fasting on days 1 and 2 of the estrous cycle. Intraperitoneal injection of 2-deoxy-D-glucose (2DG) was used to block both central and peripheral glucose oxidation, and intragastric treatment with methyl palmoxirate (MP) was used to inhibit peripheral long-chain fatty acid oxidation during the fasting and leptin-treatment period. 2DG or MP were administered at doses that did not induce anestrus in ad libitum-fed hamsters. Despite elevated central levels of leptin, fasting-induced anestrus occurred in hamsters treated with either 2DG or MP. Thus an elevated intracerebroventricular leptin concentration is not a sufficient condition for normal estrous cycles when fuel oxidation is inhibited. These results raise the possibility that central leptin influences reproduction by indirect effects on peripheral fuel metabolism. (+info)