摘要:

Abstract:Study of transmembrane signaling via G proteins has focused to a large extent upon investigations of individual G protein‐linked receptor‐effector systems. Agonist‐induced desensitization and down‐regulation of β‐adrenergic receptors, for example, have been studied extensively and adopted as a general model for G protein‐linked receptor regulation. This review focuses not only on agonist regulation of adrenergic receptor gene expression, but also on how agonists regulate opposing adrenergic receptor‐mediated pathways. This important feature of G protein‐mediated pathways, i.e., cross‐regulation and integration of information among several pathways, will be discussed in the context of what has been learned in the adrenergic recepto

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05816.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05817.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Murine neuroblastoma N1E‐115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E‐115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either125I‐AngII or the high‐affinity antagonist125I‐[Sarc1,Ile8]‐AngII. This apparent down‐regulation was dose related with an ED50of 1 nM, and maximal decreases of ∼90% were obtained with 100 nMAngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist‐exposed cells with non‐hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1receptor subtype during the first 5 min, followed by a decline in both AT1and AT2receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long‐term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down‐regulation of receptors, and the high‐affinity antagonist [Sarc1, Thr8]‐AngII blocked agonist‐induced down‐regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E‐115 cells. These results suggest that differentiated N1E‐115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII rec

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05818.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus‐secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10−4Macetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation‐dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 × 10−4mol/L. Ca2+did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 μM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 μMRG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size‐exclusion chromatography. The molecular mass of the enzyme as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was found to be between

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05819.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:There appear to be two anatomically distinct β‐endorphin (βE) pathways in the brain, the major one originating in the arcuate nucleus of the hypothalamus and a smaller one in the area of the nucleus tractus solitarius (NTS) of the caudal medulla. Previous studies have shown that these two proopiomelanocortin (POMC) systems may be differentially regulated by chronic morphine treatment, with arcuate cells down‐regulated and NTS cells unaffected. In the present experiments, we examined the effects of chronic opiate antagonist treatment on βE biosynthesis across different CNS regions to assess whether the arcuate POMC system would be regulated in the opposite direction to that seen after opiate agonist treatment and to determine whether different βE‐containing areas might be differentially regulated. Male adult rats were administered naltrexone (NTX) by various routes for 8 days (subcutaneous pellets, osmotic minipumps, or repeated intraperitoneal injections). Brain and spinal cord regions were assayed for total βE‐ir, different molecular weight immunoreactive β‐endorphin (βE‐ir) peptides, and POMC mRNA. Chronic NTX treatment, regardless of the route of administration, reduced total βE‐ir concentrations by 30–40% in diencephalic areas (the arcuate nucleus, the remaining hypothalamus, and the thalamus) and the midbrain, but had no effect on βE‐ir in the NTS or any region of the spinal cord. At the same time, NTX pelleting increased POMC mRNA levels in the arcuate to ∼ 140% of control values. These data suggest that arcuate POMC neurons are up‐regulated after chronic NTX treatment (whereas NTS and spinal cord systems remain unaffected) and that they appear to be under tonic inhibition by endogenous opioids. Chromatographic analyses demonstrated that, after chronic NTX pelleting, the ratio of full length βE1–31to more processed βE‐ir peptides (i.e., βE1–27and βE1–26) tended to increase in a dose‐dependent manner in diencephalic areas. Because βE1–31is the only POMC product that possesses opioid agonist properties, and βE1–27has been posited to function as an endogenous anatgonist of βE1–31, the NTX‐induced changes in the relative concentrations of βE1–31and βE1–27/βE1–26may represent a novel regulatory m

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05820.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Phosphatidylserine was labeled by incubating rat brain homogenates with [3‐14C]serine in the presence of Ca2+(base‐exchange conditions). Some labeled phosphati‐dylethanolamine also forms, in spite of the inhibition of Ca2+on phosphatidylserine decarboxylase. Phosphatidylserine labeling and decarboxylation also occur on incubating a mixture of purified mitochondria and microsomes, suggesting that no soluble factors are necessary for the synthesis and the decarboxylation of phosphatidylserine. Ca2+favors the transfer of phosphatidylserine from microsomes (where it forms) to mitochondria (where it is decarboxylated). The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmentalized in microsomes and that radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospho

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05821.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Poly(A)+mRNA was extracted from cultures of neurons isolated from mouse embryonic day 14 cerebral cortex and injected intoXenopusoocytes. This led to the expression of receptors for γ‐aminobutyric acid (GABA), glycine, acetylcholine, serotonin, glutamate, kainate,N‐methyl‐D‐aspartate, and quisqualate. Northern blot analysis of poly(A)+mRNA from the cultured neurons with aGluRIcDNA probe revealed the presence of three hybridization bands with estimated mRNA sizes of 5.1, 4.0, and 3.1 kb, respectively. The development of mRNAs coding for neurotransmitter receptors was investigated by isolating mRNA from neurons cultured for 2, 8, and 14 days in vitro and injecting it intoXenopusoocytes. The amplitude of membrane currents elicited by the transmitters gave a measure of the relative amounts of the different mRNAs. The size of the responses to kainate, aspartate (together with glycine), glutamate, acetylcholine, GABA, serotonin, and glycine increased with the time of culture in vitro. However, in contrast to all other agonist‐induced currents, the current induced by glycine failed to increase further from 8 to 14 days in culture. It is concluded that the time course of receptor development in cortical neurons in vitro is similar to the developme

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05822.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:In a global model of brain ischemia, accumulation of amino acids was studied in the extracellular space of the auditory cortex and the internal capsule using microdialysis, and in CSF of halothane anesthetized cats. In both brain regions, blood flow determined by hydrogen clearance decreased below 10 ml/100 g/min after extracranial multiple‐vessel occlusion, and extracellular potassium activity (Ke) measured in the dialysate increased significantly. A delayed rise inKewas observed in CSF. In contrast, ischemic amino acid accumulation differed markedly between the two brain regions investigated. In cortex, transmitter amino acids glutamate, aspartate, and γ‐aminobutyric acid (GABA) rose almost immediately after onset of ischemia, and increased 30‐, 25‐, and 250‐fold, respectively, after 2 h of ischemia. The nontransmitter amino acids taurine, alanine, and serine increased 10‐, seven‐, and fourfold, respectively, whereas glutamine and essential amino acids (valine, phenylalanine, isoleucine, and leucine) increased only 1.5‐fold. In the internal capsule, increases in amino acids, if any, were delayed and much smaller than in cortex. The largest alteration was a fivefold elevation of GABA. In CSF, changes in amino acids were small and comparable to those in the internal capsule. Our results demonstrate that ischemia‐induced extracellular amino acid accumulation is a well localized phenomenon restricted to gray matter structures that possess release and reuptake systems for these substances. We assume that amino acids diffuse slowly into adjacent white matter struct

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05823.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:This study attempts to determine whether the pathways from the guinea pig dorsal nucleus of the lateral lemniscus (DNLL) to the inferior colliculus (IC) use γ‐aminobutyric acid (GABA) as a transmitter. Injections of kainic acid (KA) were used to destroy neurons in the left DNLL. Two to 4 days after the injection, Nissl‐stained sections through the lesion site showed destruction of the DNLL neurons. The lesions varied in size; 12–100% of the DNLL neurons were destroyed on the injected side without damage to the ipsilateral IC. Two to 4 days after the injection, the electrically evoked, Ca2+‐dependent release and high‐affinity uptake of [3H]GABA were measured in dissected pieces of the left and right IC. These activities were compared with those in the IC taken from unlesioned controls and from sham controls, which received injections of saline instead of KA. Each IC was divided into a dorsal piece, which contained the dorsal cortex and dorsomedial nucleus, and a ventral piece, which contained the central and lateral nuclei. Lesions of the left DNLL depressed the release and uptake of [3H]GABA in the ventral pieces of the IC, but there was a greater depression in the ventral IC contralateral to the lesioned DNLL. There were good correlations between the percentage of neuronal loss in the left DNLL and deficits in [3H]GABA release and uptake activities in the ipsi‐ and contralateral ventral IC. By contrast, there was no depression of [3H]GABA release and uptake in the dorsal pieces of the IC. The localization of the deficits in release and uptake appears to match the distribution of the synaptic endings of the DNLL pathways in the IC. This correspondence associates GABA release and uptake activities with the DNLL projections to the IC and, therefore, suggests that GABA may be a transmitter of these pathways. The release and uptake of [14C]glycine was also measured to determine whether glycine might be a transmitter of the DNLL pathways to the IC. Lesions of the left DNLL failed to alter the Ca2+‐dependent release or the uptake of [14C]glycine, suggesting that DNLL neurons are unlikely to use this compound as

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05824.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D2autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high‐affinity uptake for [3H]DA. In rat striatal tissue, activation of D2receptors has been shown to inhibit the release of DA. Previously accumulated [3H]DA was released from the cultures upon depolarization in a Ca2+‐dependent manner. K+‐evoked [3H]DA release could be inhibited by the selective D2receptor agonists LY 171555 and N0437 in a concentration‐dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D2receptor antagonist sulpiride. These observations are indicative for the expression of functional D2receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase‐immunoreactive neurons, or the uptake capacity for [3H]DA. Our data demonstrate that chronic stimulation of DA D2receptors does not impair survival or differentiation of cultured fetal dopaminergi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb05825.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY