ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10030.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Histamine activation of H1receptors stimulates3H release from cultured bovine adrenal chromaffin cells preloaded with [3H]noradrenaline. The initial (1‐min) release induced by a high concentration of histamine was unaffected by the removal of extracellular Ca2+, whereas the more sustained response (10 min) was largely inhibited. In contrast, release induced by nicotine was dependent on extracellular Ca2+at all times. The protein kinase inhibitor staurosporine inhibited both the initial and sustained (10‐min) phases of histamine‐induced release (IC50in the region of 200 nM) but was ineffective against a direct depolarizing stimulus (56 mMK+). In contrast, the calmodulin antagonist trifluoperazine was equally effective against both stimuli. These data indicate that although a staurosporine‐sensitive event (perhaps involving protein kinase C) is essential for coupling histamine receptor activation to the release processes, it is not essential for exocytosis itself. A further distinction between histamine‐and depolarization‐induced release was demonstrated by the differential effect of the guanine nucleotide‐binding protein inhibitor pertussis toxin. Pretreatment with pertussis toxin (0.1 μg/ml for 16 h) enhanced depolarization‐induced release by ∼1.5‐fold. This pertussis toxin pretreatment was, however, approximately twofold as effective in potentiating histamine‐evoked release. Thus, the characteristics of the histaminergic response are distinct from those of a depolarizing stimulus, perhaps indicating the involvement of different mechanisms

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10031.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Parameters of ligand binding, stimulation of low‐KmGTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH‐SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the μ‐selective opioid agonist [3H]Tyr‐D‐Ala‐Gly(Me)Phe‐Gly‐ol ([3H]DAMGO) bound to two populations of sites withKDvalues of 3.9 and 160 nM, with<10% of the sites in the high‐affinity state. Both sites were also detected at 4°C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low‐affinity binding present. The toxin specifically reduced high‐affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high‐affinity binding component in cells. Addition of GTP to membranes reduced theBmaxfor [3H]DAMGO by 87% and induced a linear ligand binding component; a low‐affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high‐affinity opioid agonist binding represents the ligand‐receptor‐guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP. Opioid receptor coupling to adenylate cyclase in intact and fragmented cells occurred with similar efficiency: DAMGO inhibited adenylate cyclase withKi, values of 11 nMin cells and 26 nMin lysates, with 30% maximal inhibition in both preparations. Receptor coupling to G protein in membranes occurred with similar parameters: DAMGO stimulated low‐KmGTPase with aKsof 31 nMand anSmaxof 48%. Both effector responses were blocked by naloxone and were strongly impaired by rigorous cell homogenization. These results indicate that opioid signal transduction in intact SH‐SY5Y cells and their appropriately isolated membranes functions with similar efficiencies involving a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10032.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:To investigate the role of mesolimbic dopamine (DA) in the mechanism of drug dependence, extracellular DA was monitored by transcerebral dialysis in the caudal nucleus accumbens under basal conditions and after challenge with morphine (5 mg/kg s.c.) in control rats and in rats made dependent on and then deprived of morphine. Withdrawal from morphine resulted in a marked reduction of extracellular DA concentrations from control values at 1, 2, 3, and 5 days of withdrawal. After 7 days of withdrawal, DA output was less, but still significantly, reduced. Challenge with morphine resulted in stimulation of DA output in controls (maximum, 35%), no effect on the first day of withdrawal, and stimulation similar to controls’ on days 2 and 7 of withdrawal. On day 5 and, particularly, on day 3 of withdrawal, morphine‐induced stimulation of DA output was markedly potentiated (maximum, 100 and 160%, respectively). Changes in the sensitivity of DA transmission to morphine challenge were associated with changes in the behavioral stimulant effects of morphine, with tolerance on day 1 and marked sensitization on days 3 and 5 but also on day 7, when morphine‐induced stimulation of transmission was no longer potentiated. The results indicate that repeated morphine administration induces a state of dependence in DA neurons and a short‐lasting tolerance followed by an increased sensitivity to its stimulant effects on DA transmission. These changes might play an important role in the development of opiate addiction and in the maintenance of opiate self‐administration in dependent

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10033.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:It has been proposed that abnormalmyo‐inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may altermyo‐inositol metabolism and content. Recently, we have shown that L‐fucose, a 6‐deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor ofmyo‐inositol transport. To examine the effect of L‐fucose onmyo‐inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L‐fucose. L‐Fucose is a competitive inhibitor of Na+‐dependent, high‐affinitymyo‐inositol transport. TheKifor inhibition ofmyo‐inositol transport by L‐fucose is about 3 mM. L‐Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L‐fucose is inhibited by Na+depletion, D‐glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neithermyo‐inositol nor L‐glucose inhibits L‐fucose uptake. Chronic exposure of neuroblastoma cells to 1–30 mML‐fucose causes a decrease inmyo‐inositol accumulation and incorporation into inositol phospholipids, intracellular freemyo‐inositol content, and phosphatidylinositol levels. Na+,K+‐ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L‐fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mMglucose. Cellmyo‐inositol metabolism and Na+/K+‐pump activity are maintained when 250 μM myo‐inositol is added to the L‐fucose‐supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mMglucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mML‐fucose. The effect of L‐fucose on cultured neuroblastoma cell properties occurs at concentrations of L‐fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L‐fu

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10034.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet‐derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF‐responsive cells. The results suggest that PDGF‐like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF‐like levels. The results are discussed with respect to the possible role of PDGF in regen

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10035.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Specific polyclonal antibodies that distinguish the two distinct isoforms of the catalytic subunit of calmodulin‐dependent protein phosphatase, calcineurin Aα and Aβ, were prepared, and the distribution of calcineurin Aα and Aβ in rat brain was studied using immunochemical and immunocytochemical techniques. Immunochemical measurement revealed that the regional distributions of the two isoforms differed and that Aα was more abundant than Aβ in the rat brain. The subcellular distribution patterns of both isoforms were similar. Both isoforms were highly enriched in cytosolic fractions, including the synaptosomal cytosol. Immunocytochemical analysis revealed that both Aα and Aβ immunoreactivities differed in regional and cellular localizations. These different patterns of expression suggest that the two isoforms of calcineurin A may each have specific functions in modulating neuronal activity in particular c

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10036.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Previous results from our laboratory suggest that long‐term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12‐myristate 13‐acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine‐synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long‐term effects. Nicotine increased particulate PKC activity in a concentration‐dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1‐dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine‐receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13‐dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic‐receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine‐stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic‐receptor activation increases PKC activity and immunoreactivity in BAM cells. The long‐term PKC activation may serve several functions, such as activation of mRNA production and a negative feedback regulation of either nicotinic receptors or volta

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10037.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin‐like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5‐day‐old nerve revealed that P0contained predominantly endo‐β‐β‐acetylglucosa‐minidase H (endo H)‐resistant, complex‐type oligosaccharide. In contrast, P0from adult rats had mostly endo H‐sensitive carbohydrate, indicating the presence of appreciable high‐mannose and/or hybrid‐type oligosaccharide on the glycoprotein. The endo H‐sensitive and resistant P0of adult nerve could be readily phosphorylated by protein kinase C, as could the complex‐type P0from 5‐day‐old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of35SO42‐labeled P0showed that the sulfate group was found on both endo H‐sensitive and ‐resistant oligosaccharide. The endo H‐sensitive P0carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose‐labeled P0glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0from adult nerve to interact withGalanthus nivalisagglutinin. The observed age‐dependent changes of P0oligosaccharide may modify the b

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10038.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:To determine the phosphorylation sites of the τ in paired helical filaments (PHF), two types of PHF antisera with different specificities were used: One was a conventional anti‐PHF, and the other was an antiserum to formic acid‐denatured PHF (anti‐HFoPHF). Phosphorylated τ‐specific antibodies, anti‐ptau 1 and anti‐ptau 2, were prepared from anti‐PHF and anti‐HFoPHF, respectively. We found that both anti‐ptau 1 and anti‐ptau 2 labeled fetal or juvenile τ but not adult τ. The anti‐ptau 1‐ and anti‐ptau 2‐recognition sites were immunochemically localized to the fragment Asp313to Ile328in the most COOH‐terminal portion of τ. Furthermore, Ser315was determined as the anti‐ptau 2 recognition site. The sequence surrounding Ser315was not found in the canonical sequenc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb10039.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY