摘要:

Abstract:Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage‐sensitive Ca2+channels (VSCCs) in a cyclic AMP (cAMP)‐independent manner in PC12 cells. Ca2+influx induced by membrane depolarization with 70 mMK+was inhibited when cells were preincubated with 10 µMforskolin. Almost maximum inhibitory effect on Ca2+influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca2+influx was not affected by the presence of 2′,5′‐dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin‐induced cAMP production. 1,9‐Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca2+influx induced by 70 mMK+without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010083.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Retinoic acid (RA) produced a dose‐dependent inhibition of PC12 cell growth and the appearance of cell clusters without neurite extension. RA‐induced cell clumping was similar to that caused by dexamethasone (Dx). Nerve growth factor (NGF) induced neurite extension, and the combination of RA plus NGF produced a maximal decrease in cell proliferation with a mixed morphology in which part of the cell population had neurites and part formed clumps. Transcriptional effects of RA were demonstrated by the increase in the activity of reporter constructs that contain an RA response element. RA also regulated expression of endogenous genes in PC12 cells. The retinoid produced a two‐ to threefold increase in level of p75LNGFRmRNA (the low‐affinity NGF receptor), without altering expression of thetrkprotooncogene (the high‐affinity NGF receptor carrying tyrosine kinase activity). RA also caused a transient increase in level of tyrosine hydroxylase (TH) mRNA (twofold after 16 h), which returned to basal levels and then decreased relative to basal levels at 48 h. The effect of NGF on the expression of these genes was identical to that produced by RA. However, incubation with Dx did not induce p75LNGFRmRNA and produced a strong and sustained increase of TH mRNA level (three‐ to fivefold after 48 h). These results show that, despite the common morphological changes produced by RA and glucocorticoids in PC12 cells, the biochemical changes caused by RA are similar to those produced by NGF. Therefore, RA could initiate a biochemical program of neuronal differentiation in PC12 cells, although a fully differentiated phenotype with neurite extension is n

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010089.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+and Na+into the neuron. To maintain Na+homeostasis, the excess Na+entering through the ion channel should be removed by Na+,K+‐ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na+,K+‐ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate‐induced activation of Na+,K+‐ATPase was dose dependent: It was appreciable (37%) at 0.1 µMand peaked (85%) at 100 µM. The increase in Na+,K+‐ATPase activity by glutamate was prevented by MK‐801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12‐myristate 13‐acetate, an activator of protein kinase C, indicating that activation of Na+,K+‐ATPase is due to decreased phosphorylation by protein kinase C. W‐7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na+,K+‐ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K+‐ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to ac

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010099.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K‐252a, H‐7, and H‐8) and reportedly selective protein kinase inhibitors (KN‐62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and45Ca2+uptake. Treatment of chromaffin cells with 10 µMK‐252a, 50 µMH‐7, or 50 µMH‐8 induced changes in cell morphology within 3 h; these compounds also induced a time‐dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid‐dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN‐62, a selective inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50value of 0.32 µM), but had no effect on cell morphology. The reduction of secretory function induced by 1 µMKN‐62 was significant within 5 min and rapidly reversible. Unlike H‐7, H‐8, and staurosporine, KN‐62 significantly inhibited stimulated45Ca2+uptake. KN‐04, a structural analogue of KN‐62 that does not inhibit CaMK II, inhibited stimulated45Ca2+uptake and catecholamine release like KN‐62. These studies indicate that KN‐62 inhibits secretory function via the direct blockade of activated Ca2+influx. The nonselective inhibitors, K‐252a, H‐7, H‐8, and staurosporine, inhibit secretory function by another mechanism, perha

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010105.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In addition to its well‐known synaptic function, acetylcholinesterase was recently shown to stimulate neurite outgrowth from cultured chick neurons in a manner unrelated to its catalytic activity. It remained unclear, however, whether each of the variant acetylcholinesterase enzyme forms can promote such process extension and whether this effect of acetylcholinesterase was limited to neurite outgrowth. Using DNA microinjections and stable transfections of cultured glioma cells, we explored the possibility that specific acetylcholinesterase isoforms affect cellular development and morphology of CNS astrocytes. Cells microinjected with human ACHEDNA constructs that differ in their exon‐intron composition displayed rapid yet stable induction of cell body enlargement and process extensions. Cells transfected with ACHEDNA carrying the neuronal‐characteristic 3′‐E6 domain also displayed stable process extensions. However, stable transfections with ACHEDNAs including the 3′‐alternative I4/E5 region induced the appearance of small, round cells in a dominant manner. This was associated with expression of I4/E5‐ACHEmRNA transcripts and the production of soluble acetylcholinesterase monomers that were catalytically indistinguishable from the 3′‐E6 enzyme but displayed higher electrophoretic mobility than that of the 3′‐E6 form. Thus, variable expression levels and alternative splicing modes of the ACHE gene correlated in these experiments with glial development in a manner that was apparently

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010114.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To study how growth factors affect stimulus‐secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP‐induced [Ca2+]irise and dopamine secretion in PC12 cells. After a 4‐day incubation of cells, all three factors increased ATP‐induced dopamine secretion significantly. We then examined which step of ATP‐induced secretion was affected by the growth factors. Cellular levels of dopamine‐β‐hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP‐induced [Ca2+]irise was also enhanced after growth factor treatment. The EC50of ATP for inducing [Ca2+]irise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca2+]iof dopamine secretion was increased significantly only in NGF‐treated cells. Our results suggest that for EGF‐ and insulin‐treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca2+]irise. NGF treatment not only increased the potency of ATP but also decreased the Ca2+sensitivity of the secretory pathway, which as a result becomes more tightly regulated

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010124.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [3H]dopamine‐loaded rat striatal slices. Our results show that illumination of RBS‐pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µM), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetra(acetoxymethyl) ester (BAPTA‐AM; 10 µM) had no effect on the dopamine release stimulated by illumination of RBS‐pretreated slices. The concentration of BAPTA‐AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µM). Superfusion with zaprinast (10 µM) had no effect on RBS‐induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro t

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010131.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO‐induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold‐induced rise in TH mRNA levels. In this study, the IMO‐induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress‐elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO‐induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during I

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010138.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:5‐Lipoxygenase‐activating protein (FLAP) is an 18‐kDa integral membrane protein required, in peripheral cells, for the activation of 5‐lipoxygenase (5‐LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase‐polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5‐LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5‐LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK‐886 (0.25–1 µM) prevents the somatostatin‐induced augmentation of the hippocampal K+M‐current. Our results provide necessary evidence for the presence and signaling role of 5‐LO and FLAP in central neurons and strongly support their proposed participation in somatostatin‐rece

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010147.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed thatl‐homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium‐free medium,d‐homocysteate was slightly more potent than thel‐isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response tol‐homocysteate. 2‐Amino‐7‐phosphonoheptanoate, a potent NMDA antagonist, preferentially blockedl‐homocysteate evoked responses. 6,7‐Dinitroquinoxaline‐2,3‐dione, a potent kainate antagonist, preferentially blockedd‐homocysteate‐evoked responses. Therefore, in the rabbit retina,l‐homocysteate is an NMDA‐preferring agonist, whereasd‐homocysteate is a kainate‐preferring agonist. In addition, we found thatl‐homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA‐receptor activation. However, the low potency ofl‐homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role ofl‐homocysteate at the b

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010153.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY