Glycosyltransferase Activities in Cultured Endothelial Cells of Bovine Brain Microvascular Origin

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.     

运用植块法培养脑微血管内皮细胞

探讨简易可行的脑微血管内皮细胞(brain microvascular endothelial cells,BMECs培)养方法,为研究BMECs细胞在脑血管疾病中的重要作用提供技术支持。分离出生后1~7天内的SD乳鼠大脑皮质区,植块法培养BMECs细胞。用倒置显微镜观察BMECs细胞的形态以及从皮质块迁出的过程;MTT比色法检测BMECs细胞的生长曲线;采用免疫组化染色检测VIII因子相关抗原和CD34抗原,以鉴定内皮细胞。结果发现,大脑皮质块植块法培养的大鼠BMECs细胞呈单层贴壁生长,细胞形态以长梭形、多角形三角形、四边形为主,呈典型的“铺路石”样征象,经鉴定为内皮细胞,第三代纯度达95%以上。提示该方法具有经济、简便、要求条件不高,易于纯化的优点,可作为大鼠BMECs细胞体外培养的良好模型。     

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.     

大鼠脑微血管内皮细胞培养及其药物转运体Oatp2和P-gp的表达

贴块法培养脑微血管内皮细胞(BMECs),倒置显微镜动态观察细胞生长及形态,Ⅷ因子相关抗原、CD34免疫细胞化学联合鉴定细胞并确定纯度。免疫细胞化学和Western印迹法检测药物转运体有机阴离子转运多肽亚型2(Oatp2)及P-糖蛋白(P-gp)在培养内皮细胞上的表达。结果显示,获得的BMECs呈多角形或铺路石形,单层贴壁生长;培养细胞Ⅷ因子相关抗原免疫细胞化学、CD34免疫荧光染色均为阳性,细胞纯度90%;培养细胞有Oatp2及P-gp表达,且二者均主要表达于BMECs细胞膜。提示贴块法可获得原代培养BMECs,方法简便易行,细胞纯度较高。原代培养的BMECs上有药物转运体Oatp2及P-gp的表达,为血脑屏障上药物转运体的体外研究提供了可能途径。     

大肠杆菌yijP侵袭蛋白诱导人脑微血管内皮细胞凋亡

为研究大肠杆菌的脑微血管内皮细胞侵袭基因yijP的功能,将yijP基因（1．04kb）克隆到pQE30表达载体,构建表达产物为N末端带有6个组氨酸（His）序列的yijP汇合蛋白,以M15（pREP4）为受体菌,大量表达（His）6-yijP汇合蛋白,利用Ni—NTA亲和层析纯化汇合蛋白,将经透析法复性的一定浓度的（His）6-yijP蛋白加入到体外培养的人脑微血管内皮细胞中,结果显示yijP蛋白对人脑微血管内皮细胞有较强的细胞毒作用：在相差显微镜下可观察到细胞皱缩、胞膜呈泡状膨出,随着时间延长细胞逐渐脱落;荧光显微镜下可见细胞核呈现为致密团块状或圆形浓染颗粒状,呈凋亡样改变：DNA琼脂糖凝胶电泳可见DNA阶梯状条带;流式细胞仪显示在正常二倍体峰之前出现一个亚二倍体峰;Western印迹可检测到caspase-3的活性片段。这些现象均出现在yijP蛋白作用于人脑微血管内皮细胞的16h之后,提示在大肠杆菌侵袭人脑微血管内皮细胞过程中,yijP蛋白可能起到诱导脑微血管内皮细胞迟发性凋亡的毒素作用。     

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.     

Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Gangliosides Prevent Ischemia-Induced Down-Regulation of Protein Kinase C in Fetal Rat Brain

Complete obstruction of the maternal blood flow to fetal rats at 20 days of gestation for a period of 10 min causes a significant shift of approximately 22% in protein kinase C (PKC) activity from a cytosolic to a membrane-bound form in the fetal brain. This translocation can be entirely reversed without losses in activity by a single intraperitoneal injection into the gravid rat of either a mixture of disialo- and trisialoganglioside [polysialoganglioside (PSG)] or by GM1 (50 mg/kg of body weight) given 3 h before onset of the ischemic episode. Cessation of blood flow for 15 min followed by a reperfusion period of 24 h results in a 47% loss in total PKC activity. This down-regulation can be almost entirely prevented upon intraperitoneal administration of GM1 3 h before, but also during and even 90 min after the onset of ischemia. The PSG mixture is also effective, particularly when given 3 h before the insult. Down-regulation of PKC is accompanied by an increase in a Ca2(+)-phosphatidylserine-independent kinase [protein kinase M (PKM)] activity, which rises from 30 pmol/min/mg of protein in control animals to a maximal value of 83.1 pmol/min/mg of protein after 15 min of ischemia and 6 h of reperfusion. By 24 h, PKM activity is 46.8 pmol/min/mg of protein. Administration of GM1 blocks completely the appearance of PKM, a result suggesting that PKC down-regulation and PKM activity elevation are intimately associated events and that both are regulated by GM1 ganglioside.     

Characterization of the Scavenger Receptor on Bovine Cerebral Endothelial Cells In Vitro

Abstract— Primary cultures of bovine brain capillary endo-thelial cells (BCEC), possessing tight junctions and high levels of γ-glutamyl transpeptidase, were used as an in vitro model for the blood-brain barrier. The interaction of acetylated low density lipoprotein (AcLDL) with BCEC was studied to characterize the scavenger receptor on these cells. A saturable high affinity binding site was found with a dissociation constant of AcLDL of 5.4 μg/ml (3.1 n M ) and a maximal binding ranging from 284 to 626 ng of AcLDL/mg of cell protein for eight primary cultures, and independent of the presence of calcium. Cell association was coupled to degradation, and both could be effectively competed for by polyinosinic acid and AcLDL but not by low density lipoprotein or by high density lipoprotein. Prolonged incubation showed an accumulation of the ligand in the cells. The rate of degradation of AcLDL was ∼ 10–20-fold lower in BCEC than that of peripheral endothelial cells. No evidence for lysosomal degradation could be obtained. Binding of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocar-boxyamine perchlorate-labeled AcLDL by BCEC was observed, which could be competed for by an excess of un-labeled AcLDL and polyinosinic acid. We have shown that in vitro BCEC possesses specific binding sites for AcLDL, whereas these cells show a relatively low degradative capacity.     

蛋白激酶C对血管内皮细胞锚蛋白、CD44表达及亚细胞分布的影响

通过外源性底物对[γ-32P]-ATP的摄入量来测定豆蔻酰佛波醇乙酯(phorbol-myristate-acetate,PMA)处理后的人脐静脉内皮细胞(humanumbilicalveinendothelialcells,HUVECs)膜蛋白激酶C(proteinkinaseC,PKC)的活性;利用间接免疫荧光标记和Western印迹方法分析蛋白激酶C活性对锚蛋白及CD44的亚细胞分布及蛋白质表达的影响。结果发现HUVECs的锚蛋白及CD44表达水平趋势与PKC活性变化相吻合;PKC活化导致CD44在细胞膜上呈聚集状,而锚蛋白则移位并聚集于CD44处;PKC抑制剂能抑制PKC活化所带来的上述作用。结果表明PKC活化通过磷酸化作用能上调锚蛋白及CD44表达,并同时导致二者发生一致性运动及共分布。     

机械力对鼠脑微血管内皮细胞膜电流的影响

采用膜片钳技术以全细胞方式在鼠脑微血管内皮细胞中记录到一延迟外向电流,对Ｋ＾＋具有高度特异性,胞外施加２０ｍｍｏｌ／Ｌ的ＴＥＡ－Ｃｌ在明显抑制该电流。实验的保持电位定在－１００ｍＶ,测试电位从－１００ｍＶ至＋９０ｍＶ,每次增加１０ｍＶ,刺激波宽为２１００ｍｓ。该电流具有ＴＥＡ敏感,并有浓度依赖性,其ＩＣ５０约为２．０ｍｍｏｌ／Ｌ,类似延迟整流性钾电流特征（ＩＫｖ）。机械力作用下可引出一外向电流,膜     

A Novel Brain Neurovascular Unit Model with Neurons,Astrocytes and Microvascular Endothelial Cells of Rat

A novel triple cell neurovascular unit (NVU) model co-culturing with neurons, brain microvascular endothelial cells (BMECs) and astrocytes was established in this study for investigating the cerebral diseases and screening the candidates of therapeutic drug. We have first performed the cell identification and morphological characterization, analyzed the specific protein expression and determined the blood-brain barrier (BBB) function of the co-culture model under normal condition. Then, we further determined the BBB function, inflammation, cell injury and the variation of neuroprotective factor in this model after anoxia-reoxygenation. The results suggest that this model exhibited a better BBB function and significantly increased expression of P-glycoprotein (Pg-P) and ZO-1 compared with BMECs only or co-culture with astrocytes or neurons. After anoxia-reoxygenation, the pathological changes of this model were basically resemblance to the pathological changes of brain cells and BBB in vivo. And nimodipine, an antagonist of calcium, could reverse those changes as well. According to our observations, we deduce that this triple cell co-culture model exhibits the basic structure, function and cell-cell interaction of NVU, which may offer a more proper in vitro system of NVU for the further investigation of cerebral diseases and drug screening.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.     

Increase of Adenylate Kinase Isozyme 1 Protein During Neuronal Differentiation in Mouse Embryonal Carcinoma P19 Cells and in Rat Brain Primary Cultured Cells

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.     

人参皂苷Rb1调节脑微血管NOS/NO改善脑细胞膜通透性

目的:探究人参皂苷Rb1在脂多糖(LPS)诱导的人脑微血管细胞模型中对脑细胞膜通透性的影响。方法:采用噻唑蓝比色法(MTT)及乳酸脱氢酶(LDH)释放检测不同浓度人参皂苷Rb1对LPS刺激人脑微血管内皮细胞系(HBMEC)存活率的影响;采用EVOM法检测HBMEC细胞通透性;采用Griess法、ELISA法及DAF-FM DA荧光探针分别检测NO、ONOO~-含量及eNOS、iNOS活性;Western blot法检测p-eNOS(ser1177)、iNOS蛋白的表达水平。结果:与正常对照组比较,LPS(5μg/m L)可明显降低HBMEC细胞存活率(P0.01);与LPS组比较,随着人参皂苷Rb1浓度的增加细胞存活率明显升高,且中、高剂量组(40、80μmol/L)具有显著性差异(P0.05、P0.01);LPS导致HBMEC单层细胞电阻值明显降低(P0.01),人参皂苷Rb1高剂量组(80μmol/L)可显著抑制LPS所致的细胞膜通透性升高(P0.05);LPS可明显降低HBMEC细胞NO含量(P0.01)升高ONOO~-含量(P0.05),人参皂苷Rb1中、高剂量组(40、80μmol/L)较LPS组具有显著升高NO含量,降低ONOO~-含量的作用(P0.05);与正常对照组比较,LPS组可明显降低HBMEC细胞磷酸化eNOS (ser1177)蛋白的表达水平,而显著升高iNOS蛋白表达水平(P0.01)。人参皂苷Rb1高剂量组(80μmol/L)较LPS组可显著升高p-eNOS(ser1177)蛋白的表达水平,降低iNOS蛋白的表达水平(P0.05)。结论:人参皂苷Rb1可有效降低iNOS活性,升高eNOS活性继而减少NO水平及ONOO~-含量,显著降低LPS导致的脑细胞膜通透性升高。     

Cultured Astrocytes Release a Factor that Decreases Endothelin-1 Secretion by Brain Microvessel Endothelial Cells

Abstract: Endothelin-1 (ET-1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood-brain barrier, have been shown to secrete ET-1, whereas specific ET-1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET-1 and its precursor big ET-1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET-1 production was purified by gel-exclusion and ion-exchange chromatography as a 280-Da iron-containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron-nitrogen oxide complex, may be involved in a regulatory loop of ET-1 production at the level of the blood-brain barrier.     

miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后,将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养,其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖,TUNEL染色检测BMVEC凋亡,Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较,agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较,antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较,O/H组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe²⁺含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较,O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高,细胞活力升高,TUNEL阳性率和Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高,迁移数量升高,SOD和GSH-Px活性升高,MDA含量降低,Fe²⁺含量和PTGS2的蛋白表达水平降低,GPX4的蛋白表达水平升高,MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较,O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe²⁺含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。     

Long-Term Activation of Protein Kinase C by Angiotensin II in Cultured Bovine Adrenal Medullary Cells

Previous studies from our laboratory suggest that protein kinase C (PKC) is involved in the angiotensin II (AII)-induced increase in the expression of genes encoding proenkephalin and catecholamine biosynthesizing enzymes in primary cultured bovine adrenal medullary (BAM) cells. The purpose of this study was to examine the effects of [Sar1]-AII (S1-AII), an AII agonist, on PKC activity in BAM cells. Thirty-minute incubation with S1-AII produced a dose-dependent activation of PKC. The particulate PKC activity was significantly increased by 2 nM S1-AII after both 30 min and 12 h of incubation. A high concentration of S1-AII (200 nM) caused an increase in particulate PKC activity after 30 min of incubation and this increase was still observed after 18 h of continuous incubation. [Sar1, Thr8]-angiotensin II (S1, T8-AII) (100 microM), an AII antagonist, inhibited the effect of S1-AII (20 nM) on PKC activity, suggesting a specific AII receptor-mediated effect. An increase in BAM cell particulate PKC immunoreactivity after 18 h of S1-AII treatment was observed in Western blot analysis of PKC-immunoreactive protein (82 kDa). The persistent activation of PKC seen in this study is consistent with our hypothesis that PKC may mediate the S1-AII-induced increase in the expression of genes encoding proenkephalin and catecholamine synthesizing enzymes in BAM cells.