摘要:

Abstract: Subcutaneous administration of methylmercuric chloride to neonatal rats resulted in movement and postural disorders during the fourth postnatal week. Sodium‐dependent high‐affinity uptake of radiolabeled choline, glutamate, and γ‐aminobutyric acid (GABA) was measured in homog‐enates of cerebral cortex and caudate‐putamen. There was a significant decrease in the uptake of [3H]choline in the cerebral cortex, but not in the caudate‐putamen, at the onset of neurological impairment (73‐75%) and at one subclinical stage of toxicity (58‐64%). No significant differences in [3H]glutamate uptake were detected in either region. The uptake of [3H]GABA in the presence of 1 mMβ‐alanine, which was employed to inhibit the glial uptake process, was reduced significantly in both the cerebral cortex and caudate‐putamen at the onset of neurological impairment (50‐62%) and at one subclinical stage (40‐51%). This decrease in [3H]GABA up‐take is consistent with the results of previous studies using this animal model, which demonstrated a preferential degeneration of GABAergic neurons in the cerebral cortex and caudate‐putamen of methylmercury‐treated animals. Because the high‐affinity uptake of choline is the rate‐limiting step for acetylcholine synthesis by cholinergic neurons, the decrease in [3H]choline uptake may reflect an abnormal development of cholinergic

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07386.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3‐methoxy‐4‐hydroxyphenylethylene glycol (MHPG) and normetane‐phrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3‐methoxytyramine (3‐MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3‐MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood‐brain barrier, CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug‐naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3‐MT to define their correlational relationships. For the NE me‐tabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806,p<0.001), plasma MHPG and CSF NMN (r= 0.753,p<0.001), and plasma and CSF MHPG (r= 0.776,p<0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady‐state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3‐MT correlating with plasma HVA (r= 0.301,p<0.025). The results for the DA metabolites probably reflect regional differences in steady‐state br

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07387.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Blood‐brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB‐specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16‐18K triplet of low‐molecular‐weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary‐free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16‐18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and C4 reverse‐phase HPLC. Amino acid sequencing of the N‐terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are α‐globin, histone 2B, histone 3, and histone 2A, respectively. SDS‐PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16‐18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14‐18K proteins seen on SDS‐PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary protei

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07388.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: The β1and β2‐adrenoceptor populations in rat cortex were individually quantified by labelling all of the receptors with [3H]dihydroalprenolol and displacing with iso‐prenaline (200 μM) or CGP 20712A (l‐{2‐[(3‐carbamoyl‐4‐hydroxy)phenoxy]ethylamino}‐3‐[4‐(l‐methyl‐4‐trifluoro‐methyl‐2‐imidazolyl)phenoxy]‐2‐propanol methanesul‐phonate; 100 nM) to define total β‐adrenoceptors and β1‐adrenoceptors, respectively. Binding parameters for β2‐adrenoceptors were calculated by the difference. Oral administration of the monoamine reuptake inhibitors sibutramine HC1 (3 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg), or zimeldine (10 mg/kg) for 10 days decreased the total number of β‐adrenoceptors present in rat cortex. This effect was entirely due to a reduction in the number of β1‐adrenoceptors. Similarly, 10 days of treatment with the monoamine oxidase inhibitor tranylcypromine (10 mg/kg p.o.) or five electroconvulsive shocks (ECSs; 200 V, 2 s) spread over this period also down‐regulated β‐adrenoceptors by reducing the content of the βsubtype. By contrast, treatment with clenbuterol (5 mg/kg p.o.) for 10 days reduced the number of cortical β‐adrenoceptors by an effect on the β2‐adre‐noceptor population. The effects of short‐term treatment with these drugs were also investigated, and, using the doses shown above, the results of 3 days of administration or a single ECS were determined. Sibutramine HC1 and desipramine were alone in producing a reduction in number of β‐adrenoceptors after 3 days. Once again, this was exclusively due to a loss of β1‐adrenoceptors. Together, the results show that antidepressants with disparate pharmacological actions all down‐regulated β‐adrenoceptors through the neuronal β1‐adrenoceptor subtype. In addition, sibutramine HC1 and desipramine produced this attenuation very rapidly. However, clenbuterol treatment reduced the number of β2‐adrenoceptors, and its re

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07389.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Inositol hexakisphosphate (InsP6) increased45Ca2+uptake in cultured cerebellar granule cells. This increase was concentration dependent (EC50= 20 μM), exhibited slow kinetics, and was present after 5 days of cell maturation in culture. InsP6also enhanced D‐[3H]aspartate release in cerebellar granule cells at 11‐12 days in vitro. Stimulation of45Ca2+uptake was also produced by inositol pentakisphos‐phate but not by inositol 1,3,4,5‐tetrakisphosphate. The increase in45Ca2+influx induced by InsP6was independent of extracellular Na+and was only partially reduced by the organic calcium channel blocker nifedipine. The intrinsic action of InsP6was not affected by competitive or noncompetitive glutamate receptor antagonists. In addition, stimulations of45Ca2+uptake by InsP6and glutamate were additive. These data provide evidence that InsP6directly activates a specific population of neurons in

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07390.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Isolated rat brain capillaries were incubated in the presence of high‐density lipoprotein (HDL) containing[stearic acid‐14C, (methyI‐3H)choline]sphmgomyelim. This double‐labeled sphingomyelin was taken up in a concentration‐dependent manner. Cerebral capillary‐associated sphingomyelin had a3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hy‐drolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of in‐corporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of “pulse‐chase” experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidylc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07391.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Competitive binding studies indicated that PC 12 cells have receptors for insulin‐like growth factor‐I (IGF‐I). There are ∼ 11,100 ± 1,500 IGF‐I receptors/cell; these receptors have an apparentKDfor IGF‐I of 7.2 ± 0.6 nM.Covalent cross‐linking ofl25I‐IGF‐I to PC 12 cells labeled a 125,000‐130,000‐Mrprotein, presumably the α‐subunit of the IGF‐I receptor. Although PC 12 cells also have insulin receptors, the125I‐IGF‐I appeared to be cross‐linked to IGF‐I receptors, because 100 nMIGF‐I competed for labeling but 100 nMinsulin did not. Bovine chromaffin cells also have IGF‐I receptors. The protein tyrosyl kinase activity of IGF‐I receptors from bovine adrenal medulla and PC 12 cells was examined after purification of the receptors by wheat germ agglutinin‐Sepharose chromatography. IGF‐I (10 nM) stimulated autophosphorylation of the β‐subunits of the IGF‐I receptors from both preparations; the β‐subunits from both sources had Mrvalues of ∼97,000. IGF‐I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF‐I (IC50of ∼0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF‐I (10 nM) increased phosphorylation approximately threefold. IGF‐I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC 12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM.This result probably reflects the presence of comparable numbers of IGF‐I and insulin receptors on PCI 2 cells. Our data indicate that the IGF‐I receptors on PC 12 cells are similar to IGF‐I receptors in the adrenal medulla and other tissues. Presumably these receptor

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07392.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense‐core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin‐binding protein (65‐CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65‐CMBP and synaptophysin). Subsequent im‐munoblotting and125I‐Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65‐CMBP was present in fractions corresponding t o granule membranes and intact chromaffin granules. Similar immunoblotting and125I‐Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti‐synaptophysin and high125I‐Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65‐CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65‐CMBP and synaptophysin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07393.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: Purified adrenomedullary plasma membranes contain two high‐affinity binding sites forl25I‐ω‐conotoxin, withKDvalues of 7.4 and 364 pMandBmaxvalues of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin‐receptor complex, with at1/2of 81.6 h for the slow dissociation component. Unlabeled ω‐conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two‐site model withKi1of 6.8 andKi2of 653 pM.Specific binding was not affected by Ca2+channel blockers or activators, cho‐linoceptor antagonists, adrenoceptor blockers, Na+channel activators, dopaminoceptor blockers, or Na+/H+antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration‐dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with aKDof 490 pMand aBmaxof 129 fmol/mg of protein. At 0.25 μM, co‐conotoxin was notable to block depolarization‐evoked Ca2+uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mMK+for 30 s, whereas under the same conditions, 1 μMnitrendipine inhibited uptake by ∼60%. When cells were hyper‐polarized with 1.2 mMK+for 5 min and then Ca2+uptake was subsequently measured during additions of 59 mMK+, ω‐conotoxin partially inhibited Ca2+uptake in a concentration‐dependent manner. These results suggest that two different types of Ca2+channels might be present in chromaffin cells. However, the molecular identity of ω‐conotoxin bin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07394.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract: The involvement of the γ‐aminobutyric acidA(GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide‐treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15‐1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide‐treated rats. The inhibition of [3H]Ro 15‐1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAAreceptor complex in well‐washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition‐site qualities of the constituent proteins of the GABAAreceptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb07395.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY