Molecular level investigation of exocytosis in cultured human pheochromocytoma cells: insights from high resolution scanning electron microscopy combined with autoradiogram and cytochemistry.
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OBJECTIVE: To investigate molecular events of exocytosis in cultured human pheochromocytoma cells with stimulation. METHODS: The cultured pheochromocytoma cells prepared from human adrenal pheochromocytoma tumor were stimulated for the release of catecholamines by depolarization with the administration of 50 mmol/L KCl. Transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HR-SEM) combined with autoradiography and cytochemistry were used to observe molecular mechanisms of exocytotic release of catecholamines from the stimulated cells labelled with 3H-noradrenaline and the filipin-treated cells. RESULTS: TEM and HR-SEM observations of the stimulated cells labelled with 3H-noradrenaline revealed that the initial exocytotic fusion pores even less than 10 nm in diameter in human pheochromocytoma cells can be clearly observed in a single lipid bilayer. Furthermore, HR-SEM examinations of the filipin-treated cells showed that the derangement of the particles of the filipin-sterol complexes (FSCs) in the fused membranes of granule and plasma membranes occurred as the exocytotic fusion pores opened. In addition, the aggreates of the FSCs particles were consistently demonstrated around the openings of the differently sized closing exocytotic pores. CONCLUSIONS: Based on our results, it is suggested that the rearrangement of the sterol molecules in the fused membranes of granule and plasma membranes plays an important role in the opening and closing mechanisms of exocytotic fusion pores. We hope that morphological data obtained in this study can provide some new insights into the understanding of molecular mechanisms of exocytosis, particularly the opening and closing of exocytotic fusion pores in relation to the distribution of the membrane sterols. (+info)
The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli.
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Studies were performed to determine if the unique N-terminal domain of the R1 subunit from herpes simplex virus (HSV) type 1 ribonucleotide reductase is a substrate for casein kinase 2 (CK2). Transphosphorylation assays demonstrated that R1 was highly phosphorylated by this enzyme with multiple phosphorylation sites mapped to the N terminus between residues 1 and 245. Immunoprecipitation pull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1 and CK2 but residual amounts of CK2 present after immunoprecipitation efficiently transphosphorylated R1. Activity assays with a peptide substrate identified CK2 in R1 immunoprecipitated from infected-cell extracts but did not detect activity in R1 proteins immunoprecipitated from bacterial extracts. However, Western blotting identified potential E. coli homologues of the CK2 alpha and beta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not a protein kinase and that all previous results can be explained by contaminating kinases, principally CK2. (+info)
Hormonal regulation of natriuretic peptide system during induced ovarian follicular development in the rat.
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All components of the natriuretic peptide (NP) system have been found in the ovary. The purpose of this study was to determine the hormonal regulation of the NP system during follicular growth and ovulation induced by gonadotropins eCG and hCG. Ovarian membrane binding, before and after treatment, revealed the presence of guanylyl cyclase-type receptors exclusively. Equine CG treatment increased Bmax from 225 +/- 50 fmol/mg protein in control animals to 354 +/- 51 fmol/mg protein, and additional hCG treatment increased it further to 492 +/- 130 fmol/mg protein (p < 0.05), without changing receptor affinity. The increased binding was consistent with increased ability of atrial natriuretic peptide (ANP) to activate guanylyl cyclase in the ovarian cells obtained from hormone-treated animals. In confirmation, autoradiography of 125I-tyroCNP and 125I-ANP binding to the rat ovary showed that both guanylyl cyclase GC-A and GC-B receptor subtypes are localized to the granulosa cells of antral follicles. Quantitative analysis of GC-A and GC-B receptors by reverse transcription-polymerase chain reaction showed that the expression level of both receptors started to increase at 2 h and reached maximal levels at 6 h following eCG treatment. Increased levels of GC-B mRNA were also observed 12 h after eCG injection. At 24 and 48 h the receptor levels were below basal. Stimulation of NP receptors by eCG was paralleled by activation of both ovarian ANP and C-type natriuretic peptide (CNP) gene expression. ANP mRNA increased as early as 1 h after eCG injection and remained elevated up to 6 h. CNP mRNA increased at 2 h after eCG injection, peaked (5-fold) at 6 h, and remained elevated 48 h later, a stage at which follicular maturation continues. Incubation of ovaries with ANP significantly decreased eCG-induced estradiol level, indicating the functionality of the ovarian NP system. These results implicate the NP system in the induction and maintenance of fluid balance in the rapidly developing ovarian follicle. (+info)
Brain noradrenergic receptors in major depression and schizophrenia.
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The binding of [125I]p-iodoclonidine to alpha-2, and/or [125I]iodopindolol to beta-1 and beta-2 adrenoceptors was measured in right prefrontal cortex (Brodmann's area 10) and right hippocampus from subjects with DSM-III-R diagnoses of major depression (n = 15) or schizophrenia (n = 8) as well as from control subjects (n = 20). No significant differences between study groups were observed in binding to alpha-2 adrenoceptors in any of the six layers of prefrontal cortex or in any of the hippocampal fields. Likewise, there were no significant differences in beta-1 or beta-2 adrenoceptor binding in any of the hippocampal fields between control and major depressive subjects. In contrast, binding to beta-1 adrenoceptors, but not beta-2 adrenoceptors, was significantly lower (-13 to -27%) in most hippocampal fields of schizophrenic subjects as compared to control subjects or to major depressives. Alterations in beta-1 adrenoceptor binding in the hippocampus of schizophrenics provide further evidence for a role of central noradrenergic neurons in the neurochemical pathology of schizophrenia. (+info)
Raclopride and chlorpromazine, but not clozapine, increase muscle rigidity in the rat: relationship with D2 dopamine receptor occupancy.
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The aim of the present study was to investigate the relationship between effects on muscle tone and D2 receptor occupancy of two typical antipsychotic drugs, raclopride and chlorpromazine, and the atypical drug, clozapine. Increased muscle tone (i.e., muscle rigidity), was measured as increases in tonic electromyographic (EMG) activity of the antagonistic muscles of the rat hind limb. D2 dopamine receptor occupancy was assessed in the striatum and substantia nigra, areas involved in the regulation of muscle tone. Raclopride and chlorpromazine produced dose-dependent increases in EMG activity associated with D2 occupancy of 68%-80% in the striatum and 67%-76% in the nigra. No significant increases in EMG were observed with clozapine which showed low D2 occupancy. The results are consistent with those from human studies showing extrapyramidal side effects were associated with striatal D2 occupancy of > 70%. (+info)
Excitatory actions of NMDA receptor antagonists in rat entorhinal cortex and cultured entorhinal cortical neurons.
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We have characterized excitatory effects of non-competitive NMDA receptor antagonists MK-801, PCP, and ketamine in the rat entorhinal cortex and in cultured primary entorhinal cortical neurons using expression of immediate early gene c-fos as an indicator. NMDA receptor antagonists produced a strong and dose-dependent increase in c-fos mRNA and protein expression confined to neurons in the layer III of the caudal entorhinal cortex. Induction of c-fos mRNA is delayed and it is inhibited by antipsychotic drugs. Cultured entorhinal neurons are killed by high doses of MK-801 and PCP but c-fos expression is not induced in these neurons indicating that this in vitro model does not fully replicate the in vivo effects of PCP-like drugs in the entorhinal cortex. Excitatory effects of the NMDA receptor antagonists may be connected with the psychotropic side effects of these drugs and might become a useful model system to investigate neurobiology of psychosis. (+info)
Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse.
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine identified in the human diet and in cigarette smoke that produces prostate tumors in the rat. PhIP is bioactivated by cytochrome P-450 enzymes to N-hydroxylated metabolites that undergo further activation by conjugation enzymes, including the N-acetyltransferases, NAT1 and NAT2. To investigate the role of prostate-specific expression of human N-acetyltransferase 2 (NAT2) on PhIP-induced prostate cancer, we constructed a transgenic mouse model that targeted expression of human NAT2 to the prostate. Following construction, prostate, liver, lung, colon, small intestine, urinary bladder, and kidney cytosols were tested for human NAT1- and NAT2-specific N-acetyltransferase activities. Human NAT2-specific N-acetyltransferase activities were 15-fold higher in prostate of transgenic mice versus control mice, but were equivalent between transgenic mice and control mice in all other tissues tested. Human NAT1-specific N-acetyltransferase activities did not differ between transgenic and control mice in any tissue tested. Prostate cytosols from transgenic and control mice did not differ in their capacity to catalyze the N-acetylation of 2-aminofluorene, the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-PhIP or the N,O-acetylation of N-hydroxy-2-acetylaminofluorene. Transgenic and control mice administered PhIP did not differ in PhIP-DNA adduct levels in the prostate. This study is the first to report transgenic expression of human NAT2 in the mouse. The results do not support a critical role for bioactivation of heterocyclic amine carcinogens by human N-acetyltransferase-2 in the prostate. However, the lack of an effect may relate to the level of overexpression achieved and the presence of endogenous mouse acetyltransferases and/or sulfotransferases. (+info)
Pharmacokinetics of the 8-methoxyquinolone, moxifloxacin: tissue distribution in male rats.
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BAY 12-8039 (moxifloxacin-HCl) and 14C-labelled BAY 12-8039 were administered to male rats as single i.v. and oral doses of 4.6 and 5.0 mg/kg bodyweight respectively. The distribution of substance-associated radioactivity in the body was investigated by whole-body autoradiography. The concentrations of the unchanged compound in plasma, skin suction blister fluid and lung tissue were determined by HPLC. Whole-body autoradiography revealed distinctly higher concentrations of radioactivity in the gastrointestinal tract, urinary bladder and in most organs and tissues (e.g. kidneys, liver, spleen, lungs, various glands, cartilaginous tissues and in melanin-containing structures located in the eye, meninges and hair follicles of pigmented skin) than in blood. Radioactivity crossed the blood-brain barrier only to a small extent. The results show a high tissue affinity and a rapid and homogeneous distribution of radioactivity from blood to organs or tissues. No relevant difference in the distribution of radioactivity was found following i.v. and oral administration. After i.v. and oral dosing similar concentrations of the unchanged compound were determined in skin suction blister fluid and plasma. The concentrations of the unchanged compound in lung tissue were about three times higher than those in plasma following both i.v. and oral administration. The concentration-time courses for moxifloxacin in plasma and lung tissue were parallel. (+info)