摘要:

Abstract:Electrophysiological studies of photoreceptors from the horseshoe crabLimulus polyphemuscontinue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)+RNA isolated fromLimuluslateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) andDrosophilaphosrestin I (53% identity).Limulusarrestin was expressed in a heterologous system, and its properties were compared with those of a 46‐kDa light‐regulated phosprotein (pp46A) inLimulusphotoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca2+levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S‐antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that inLimulusphotoreceptors, light regulates the phosphorylation of arrestin in compl

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010001.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ‐opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS‐7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD= 0.23 ± 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki= 0.6 ± 0.2 nM) ≈β‐endorphin (Ki= 0.7 ± 0.5 nM) ≈ morphine (Ki= 0.8 ± 0.5 nM) ≈ [d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin (DAMGO;Ki= 1.6 ± 0.5 nM) ⋙ U50,488 (Ki= 910 ± 0.78 nM)>[d‐Pen2,5]‐enkephalin (Ki= 3,170 ± 98 nM)>dextrorphan (Ki= 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolarKiare consistent with a μ‐opioid binding site. Two additional experiments provided evidence that this opioid‐binding site is functionally coupled to G proteins: (a) In COS‐7 cells 50 µM5′‐guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50= 3.4 ± 0.5 nM) to a lower‐affinity state (IC50= 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ‐opioid receptor to DAMGO resulted in a dose‐dependent, naloxone‐sensitive inhibition of forskolin‐stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ‐opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ‐receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was a

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010014.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Dopamine β‐hydroxylase (DBH; EC 1.14.17.1) exists as membrane‐bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell‐free system. Translation of full‐length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl‐terminal, in contrast to amino‐terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site‐directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu41→ Leu41and Ser43→ Thr43yielded only the GH form with membranes, whereas mutation of Ser43→ Ala43generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting proc

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010025.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:A cDNA clone encoding a novel G protein‐linked receptor was isolated from a rat cerebral cortex cDNA library using a polymerase chain reaction‐amplified cDNA fragment as a probe. This 2.4‐kb clone encodes a 367 amino acid protein with seven putative transmembrane spanning domains. The protein is highly homologous to the cloned μ, δ, and κ opioid receptors and shares with them structural features such as three glycosylation sites in the amino terminus, a cyclic AMP‐dependent kinase phosphorylation site in the third cytoplasmic loop, an aspartic acid residue in the second transmembrane domain, and a palmitoylation site on the intracellular carboxy terminus. The receptor is also homologous with members of the somatostatin receptor family, yet it binds neither opiate nor somatostatin ligands. Northern blot analysis reveals two transcripts of 3.2 and 7.6 kb that are predominantly expressed in the cerebral cortex and hypothalamus. In situ hybridization analysis also shows a high abundance of mRNA in the cerebral cortex, hippocampus, amygdala, hypothalamus, thalamus, and dorsal raphe nuclei. It is suggested that the endogenous ligand for this receptor may represent a novel neuropeptide that may be closely related to the opiate pept

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010034.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Neurons from the granular layer of the cerebellum express functional β2‐adrenoreceptors (β2‐ARs). We show that stimulation of β2‐ARs with isoprenaline increases cyclic AMP (cAMP) production and stimulates transcription of genes containing the cAMP‐responsive element (CRE; TGACGTCA). This effect is mediated by cAMP‐dependent protein kinase and thetrans‐acting factor CRE binding protein. Transcriptional regulation by the β2‐AR was investigated by using the c‐fosprotooncogene as a model system. We show that β2‐ARs stimulate c‐fosmRNA accumulation, increase AP1binding activity, and stimulate transcription through the phorbol ester‐responsive element (TGACTCA). The transcriptional regulation of c‐fositself was studied with reporter constructs driven by c‐fospromoter sequences. Deletion studies revealed that β2‐ARs stimulate c‐fostranscription through at least three distinct regulatory sequences: (a) the CRE located at −60 bp 5′ to the initiation site, (b) the fos AP1‐like element (−291 to −297), and (c) the serum‐responsive element (−297 to −317). The regulation of these elements by the two putative second messengers of the β2‐AR, cAMP and Ca2+, was analyzed. We report that all three of these regulatory sequences are coregulated by both second messengers. These results indicate that β2‐ARs stimulate c‐fostranscription by multi

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010041.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Dopamine β‐hydroxylase (DBH) catalyzes the synthesis of the neurohormone norepinephrine and is expressed only in noradrenergic and adrenergic cells of the nervous and endocrine systems. We have previously described a positive‐acting genetic regulatory element of the DBH that contributes to the restricted expression of this gene. In the study described here, we identify and characterize a negative‐acting regulatory element within the 5′‐flanking region of the rat DBH gene. This negative regulatory element, which is located between −282 and −232, represses transcription from the DBH promoter in cell lines of noncatecholaminergic origin and binds to nuclear factors found in extracts from both the catecholaminergic cell line SHSY‐5Y and the noncatecholaminergic cell lines JEG‐3 and C6. The negative regulatory region will reduce transcription from a heterologous promoter in two noncatecholaminergic cell lines. These experiments demonstrate that the selective expression of the DBH gene is controlled by both positive‐ and negative‐acting genet

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010052.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108‐15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ∼1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′‐(γ‐thio)triphosphate>UTP = 2‐methylthio‐ATP>ADP ≫ CTP, AMP, α,β‐methylene‐ATP, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)ATP. The EC50of 1 ± 0.2 µMfor UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P2ureceptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC50= 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (IC50= 20–30 µM). Pretreatment of cells with the Ca2+ionophore ionomycin or with 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone, an inhibitor of Ca2+‐ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca2+stores, suppressed the ATP‐ or UTP‐induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca2+chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP‐induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC50for nitroarginine = 1 µM) or by hemoglobin (IC50= 2 µM). This indicates that ATP/UTP via a P2‐receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca2+level, mostly due to release of Ca2+from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross‐desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ∼40% only. This ind

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010061.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high‐affinity, hemicholinium‐3‐sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High‐affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15‐kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium‐dependent release but lacks an organized relea

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010069.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We investigated the effect of the adenosine receptor agonist 5′‐(N‐ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2bsubtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) in a time‐dependent manner. Inhibition reached 25% after 30–40‐min exposure to NECA. This effect on DMPP‐evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca2+]itransients induced by DMPP that peaked at 30‐min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+load was not affected by the treatment with NECA. Short‐term treatment with NECA failed both to modify [Ca2+]ilevels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP‐evoked Ca2+influx pathway (e.g., L‐type Ca2+channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion f

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010077.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Nitric oxide synthase (NOS) activity was induced in the cytosol of A‐172 cells by treatment with lipopolysaccharide, tumor necrosis factor‐α, and interferon‐γ. A 130‐kDa human inducible NOS (iNOS) protein was detected with anti‐rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was ∼4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A‐172 cells by reverse transcriptase‐PCR, as a probe. The mRNA was induced by interferon‐γ at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor‐α, and, to a larger extent, interleukin‐1β. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A‐172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A‐172 cells. This process requires de novo protein synthesis with a mechanism similar to that

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010085.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY