Differential effects of haloperidol and clozapine on [(3)H]cAMP binding, protein kinase A (PKA) activity, and mRNA and protein expression of selective regulatory and catalytic subunit isoforms of PKA in rat brain

The present study was undertaken to examine whether the mechanism of action of typical and atypical antipsychotics is related in their ability to regulate key phosphorylating enzyme of adenylyl cyclase-cAMP pathway, i.e., protein kinase A (PKA). For this purpose, regulatory (R) and catalytic (Cat) activities of PKA and expression of various isoforms of regulatory and catalytic subunits were examined in rat brain after single or chronic (21-day) treatment with haloperidol (HAL, 1 mg/kg) or clozapine (CLOZ, 20 mg/kg). It was observed that chronic but not acute treatment of CLOZ significantly decreased [(3)H]cAMP binding to the regulatory subunit of PKA as well as catalytic activity of PKA in particulate and cytosol fractions of the rat cortex, hippocampus, and striatum. In these fractions, CLOZ significantly decreased protein levels of selective RII alpha-, RII beta-, and Cat beta-subunit isoforms of PKA. These decreases were accompanied by decreases in their respective mRNA expression. In contrast, chronic but not acute treatment of HAL significantly increased [(3)H]cAMP binding and the catalytic activity of PKA in particulate and cytosol fractions of only the striatum brain area. In addition, chronic treatment of HAL significantly increased mRNA and protein levels of RII alpha- and RII beta-subunit isoforms in the striatum. None of the antipsychotics caused any change in the expression of the Cat alpha-, RI alpha-, or RI beta-subunit isoform. These results, thus, suggest that HAL and CLOZ differentially regulate PKA catalytic and regulatory activities and the expression of selective catalytic and regulatory subunit isoforms of PKA, which may be associated with their mechanisms of action.     

Calcium and protein kinase C (PKC)-related kinase mediate alpha 1A-adrenergic receptor-stimulated activation of phospholipase D in rat-1 cells,independent of PKC

A previous study conducted in rat-1 cells expressing alpha(1A)-adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHE-induced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not G? 6976 or a pseudosubstrate peptide inhibitor of PKCalpha, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC alpha, delta, epsilon, and eta reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by alpha1A-adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca2+ but G? 6976 and the peptide inhibitor did not. In the absence of extracellular Ca2+, PHE failed to increase PLD activity. These results indicate that alpha1A-adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKCalpha, delta, epsilon, and eta, but dependent on a PKC-related kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.     

Modifications in the phosphoinositide signaling pathway by adrenal glucocorticoids in rat brain: focus on phosphoinositide-specific phospholipase C and inositol 1,4,5-trisphosphate

The hypothalamic-pituitary-adrenal (HPA) axis has been shown to be involved in mood and behavior. The possibility that adrenal glucocorticoids regulate components of the phosphatidylinositol (PI) signal transduction pathway was investigated. Two different doses of corticosterone (CORT) pellets (50 or 100 mg) were implanted in normal and bilaterally adrenalectomized (ADX) rats, and CORT regulation of the expression of G(q) alpha protein, phospholipase C (PLC) isozymes, inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms, and of PI-PLC activity, [(3)H]IP(3) binding to IP(3)Rs, and IP(3) levels were measured in various brain areas after 1 or 14 days. Fourteen days of CORT pellet implantation into normal rats dose dependently decreased PI-PLC activity and selectively the mRNA and protein expression of PLC beta(1) isozyme in cortex and hippocampus. Bilateral ADX caused the opposite changes in these measures, and simultaneous CORT pellet implantation into ADX rats reversed these effects. Furthermore, 14 days of CORT treatment of normal rats increased [(3)H]IP(3) binding to IP(3)Rs and decreased IP(3) levels in cortex, hippocampus, and cerebellum, without any changes in expression of IP(3)R-I, IP(3)R-II, or IP(3)R-III isoform. On the other hand, ADX decreased [(3)H]IP(3) binding and increased levels of IP(3), and simultaneous CORT treatment of ADX rats prevented these changes. ADX or CORT treatment had no significant effects on the expression of G(q/11) alpha protein. These results suggest that manipulation of the HPA axis alters various components of the PI signaling pathway in rat brain, which may have physiological relevance to the HPA axis-mediated changes in mood and behavior.     

Ethanol differentially enhances hippocampal GABA A receptor-mediated responses in protein kinase C gamma (PKC gamma) and PKC epsilon null mice

Ethanol intoxication results partly from actions of ethanol at specific ligand-gated ion channels. One such channel is the GABA(A) receptor complex, although ethanol's effects on GABA(A) receptors are variable. For example, we found that hippocampal neurons from selectively bred mice and rats with high hypnotic sensitivity to ethanol have increased GABA(A) receptor-mediated synaptic responses during acute ethanol treatment compared with mice and rats that display low behavioral sensitivity to ethanol. Here we investigate whether specific protein kinase C (PKC) isozymes modulate hypnotic and GABA(A) receptor sensitivity to ethanol. We examined acute effects of ethanol on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in mice lacking either PKCgamma (PKCgamma(-/-)) or PKCepsilon (PKCepsilon(-/-)) isozymes and compared the results to those from corresponding wild-type littermates (PKCgamma(+/+) and PKCepsilon(+/+)). GABA(A) receptor-mediated IPSCs were evoked in CA1 pyramidal neurons by electrical stimulation in stratum pyramidale, and the responses were recorded in voltage-clamp mode using whole-cell patch recording techniques. Ethanol (80 mM) enhanced the IPSC response amplitude and area in PKCgamma(+/+) mice, but not in the PKCgamma(-/-) mice. In contrast, ethanol markedly potentiated IPSCs in the PKCepsilon(-/-) mice, but not in PKCepsilon(+/+) littermates. There was a positive correlation between ethanol potentiation of IPSCs and the ethanol-induced loss of righting reflex such that mice with larger ethanol-induced increases in GABA(A) receptor-mediated IPSCs also had higher hypnotic sensitivity to ethanol. These results suggest that PKCgamma and PKCepsilon signaling pathways reciprocally modulate both ethanol enhancement of GABA(A) receptor function and hypnotic sensitivity to ethanol.     

新生大鼠缺氧缺血性脑损伤蛋白激酶C与c—fps基因表达关系的探讨

目的探讨蛋白激酶C与 c-fos基因蛋白(c-Fos)表达在新生大鼠缺氧缺血性脑损伤时的作用机制.方法蛋白激酶C蛋白定量采用Lowry法;蛋白激酶C活性测定采用γ-32P催化活性测定法;c-Fos表达采用免疫组织化学染色法.结果与对照组相比,缺血、缺氧20分钟后 0、4、12、24、48、72小时及7日组新生鼠脑皮质、海马神经细胞胞膜蛋白激酶C活性降低(t=3.50,P<0.05;t=3.35,P<0.01);脑皮质神经细胞胞浆蛋白激酶C活性增高(t=4.26,P<0.01);海马神经细胞胞浆活性变化不大;上述改变在缺血、缺氧后14日组基本恢复正常.新生鼠缺血、缺氧后即刻脑组织各部分即有不同程度的 c-Fos表达,且于缺血、缺氧后4小时达高峰,以后强度逐渐降低,并持续至72小时.c-Fos表达在脑皮质以Ⅱ～Ⅴ层明显,海马以CA1、海马齿状回明显,CA3也有表达,丘脑c-Fos表达稍有增加.结论缺血、缺氧激活的蛋白激酶C可调节c-fos基因表达,持续的蛋白激酶C活性降低及c-Fos表达参与了神经细胞的损伤过程.     

新生大鼠缺氧缺血性脑损伤蛋白激酶C与c-fos基因表达关系的探讨

目的:探讨蛋白激酶C与 c-fos基因蛋白(c-Fos)表达在新生大鼠缺氧缺血性脑损伤时的作用机制.方法:蛋白激酶C蛋白定量采用Lowry法;蛋白激酶C活性测定采用γ-32P催化活性测定法;c-Fos表达采用免疫组织化学染色法.结果:与对照组相比,缺血、缺氧20分钟后 0、4、12、24、48、72小时及7日组新生鼠脑皮质、海马神经细胞胞膜蛋白激酶C活性降低(t=3.50,P<0.05;t=3.35,P<0.01);脑皮质神经细胞胞浆蛋白激酶C活性增高(t=4.26,P<0.01);海马神经细胞胞浆活性变化不大;上述改变在缺血、缺氧后14日组基本恢复正常.新生鼠缺血、缺氧后即刻脑组织各部分即有不同程度的 c-Fos表达,且于缺血、缺氧后4小时达高峰,以后强度逐渐降低,并持续至72小时.c-Fos表达在脑皮质以Ⅱ～Ⅴ层明显,海马以CA1、海马齿状回明显,CA3也有表达,丘脑c-Fos表达稍有增加.结论:缺血、缺氧激活的蛋白激酶C可调节c-fos基因表达,持续的蛋白激酶C活性降低及c-Fos表达参与了神经细胞的损伤过程.     

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.     

反义蛋白激酶Cγ寡核苷酸对原代培养大鼠脑神经元蛋白激酶Cγ mRNA表达与皮质和海马神经元增殖及神经突起生长的影响

目的:观察阳离子脂质体Lipofectamine介导的蛋白激酶CγAsODN对原代培养大鼠脑神经元蛋白激酶CγmRNA表达及生长的影响。方法:实验于2004-03/08在华中科技大学同济医学院附属同济医院完成。①实验分组:取新生50只SD乳鼠大脑皮质及海马神经元进行原代培养,分为正常对照组,空脂质体转染组,300nmol/L Lipofectamine-SODN转染组,100nmol/L Lipofectamine-AsODN转染组,300nmol/L Lipofectamine-AsODN转染组,每组10瓶。②实验方法:培养第4￣5天采用脂质体或脂质体包裹的不同浓度的AsODN或SODN蛋白激酶Cγ转染培养神经元。③实验评估:采用倒置显微镜对神经元的生长进行动态观察;免疫组化ABC法进行蛋白激酶Cγ阳性神经元的鉴定;MTT法检测神经元的增殖率;RT-PCR方法检测转染后神经元的蛋白激酶CγmRNA的表达变化。结果:①蛋白激酶Cγ阳性神经元呈胞浆内棕褐色染色,胞核不显色。②蛋白激酶CγAsODN转染早期呈现促进神经元突起生长,胞体分化的作用。随着反义浓度增加,相对生长指数值升高。而在转染晚期,ASODN蛋白激酶Cγ转染组凋亡率均明显增加。③AsODN蛋白激酶Cγ可降低神经元蛋白激酶CγmRNA的表达。结论:蛋白激酶CγAsODN可减少神经元蛋白激酶CγmRNA的表达,并刺激皮质及海马神经元的增殖及神经突起的生长,这可能与蛋白激酶Cγ对神经元的生长起负性调控作用有关。     

电针对VD大鼠海马神经细胞PKC mRNA、mGluRs和AMPAR表达的影响

目的 观察电针对血管性痴呆(VD)大鼠海马神经细胞PKC mRNA、mGluRs、AMPAR表达的影响,探讨电针的治疗作用机制.方法 将SD大鼠随机分为假手术组、模型组和电针组,每组10只.模型组和电针组采用重复脑缺血再灌注方法建立VD大鼠模型.将电针组大鼠放入大鼠固定器中,暴露大鼠头部和背部,将电针浅刺入大鼠“百会”、“大椎”穴,每天电针1次,留针20 min,连续治疗10 d.3组大鼠均于造模10d后采用逆转录聚合酶链式反应(RT-PCR)检测海马神经细胞PKC mRNA,免疫组织化学染色技术观察脑组织海马神经细胞mGluRs、AMPAR染色结果.结果 模型组海马PKC mRNA表达较假手术组降低,而与模型组比较,电针组海马PKC mRNA表达显著增加,差异有统计学意义(P＜0.05).海马mGluRs免疫阳性细胞积分光密度在假手术组、模型组和电针组分别为(58.6±3.6)、(36.3±2.5)和(51.5±4.8),与假手术组比较,模型组海马mGluRs免疫阳性细胞积分光密度显著降低,差异有统计学意义(P＜0.01);而与模型组比较,电针组海马mGluRs免疫阳性细胞积分光密度显著增加,差异有统计学意义(P＜0.05);海马AMPAR免疫阳性细胞积分光密度在假手术组、模型组和电针组分别为(66.5±2.8)、(40.1±5.1)和(58.3±4.6),与假手术组比较,模型组海马AMPAR免疫阳性细胞积分光密度显著降低,差异有统计学意义(P＜0.01),而与模型组比较,电针组海马AMPAR免疫阳性细胞积分光密度显著增加,差异有统计学意义(P＜0.05).结论 电针可增加VD大鼠海马PKC mRNA、mGluRs和AMPAR表达.电针大鼠“百会”、“大椎”穴改善大鼠学习记忆能力的机制可能与提高海马mGluRs、AMPAR和PKC mRNA表达有关.     

Preferential activation of microsomal diacylglycerol/protein kinase C signaling during glucose treatment (De Novo phospholipid synthesis) of rat adipocytes.

Glucose has been reported to increase the de novo synthesis of diacylglycerol (DAG) and translocate and activate protein kinase C (PKC) in rat adipocytes. Presently, we examined the major subcellular site of PKC translocation/activation in response to glucose-induced DAG. Glucose rapidly increased DAG content and PKC enzyme activity in microsomes, but not in plasma membranes or other membranes, during a 30-min treatment of rat adipocytes. This glucose-induced increase in microsomal DAG was attended by increases in immunoreactive PKC alpha, beta, and epsilon. Glucose-induced activation of DAG/PKC signaling in microsomes was not associated with a change in the translocation of Glut-4 transporters from microsomes to the plasma membrane, a biological response that is known to be stimulated by agonists, e.g., phorbol esters, which increase DAG/PKC signaling in plasma membranes, as well as in microsomes. In conclusion, an increase in de novo phospholipid synthesis, as occurs during glucose treatment of rat adipocytes, primarily activates DAG/PKC signaling in microsomes; moreover, this signaling response and biological consequences thereof may differ from those of agonists that primarily stimulate DAG/PKC signaling in the plasma membrane.     

Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla.

The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.     

Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation.

We compared the activity and physiologic effects of cardiac angiotensin converting enzyme (ACE) using isovolumic hearts from male Wistar rats with left ventricular hypertrophy due to chronic experimental aortic stenosis and from control rats. In response to the infusion of 3.5 X 10(-8) M angiotensin I in the isolated buffer perfused beating hearts, the intracardiac fractional conversion to angiotensin II was higher in the hypertrophied hearts compared with the controls (17.3 +/- 4.1% vs 6.8 +/- 1.3%, P less than 0.01). ACE activity was also significantly increased in the free wall, septum, and apex of the hypertrophied left ventricle, whereas ACE activity from the nonhypertrophied right ventricle of the aortic stenosis rats was not different from that of the control rats. Northern blot analyses of poly(A)+ purified RNA demonstrated the expression of ACE mRNA, which was increased fourfold in left ventricular tissue obtained from the hearts with left ventricular hypertrophy compared with the controls. In both groups, the intracardiac conversion of angiotensin I to angiotensin II caused a comparable dose-dependent increase in coronary resistance. In the control hearts, angiotensin II activation had no significant effect on systolic or diastolic function; however, it was associated with a dose-dependent depression of left ventricular diastolic relaxation in the hypertrophied hearts. These novel observations suggest that cardiac ACE is induced in hearts with left ventricular hypertrophy, and that the resultant intracardiac activation of angiotensin II may have differential effects on myocardial relaxation in hypertrophied hearts relative to controls.     

The roles of protein kinase C and tyrosine kinases in mediating endothelin-1-stimulated phospholipase D activity in rat myometrium: differential inhibition by ceramides and cyclic AMP

The aim of the present study was to investigate the mechanisms that regulate the activation of phospholipase D (PLD) by endothelin (ET)-1 in rat myometrium. We previously reported that ET-1 exerted part ( approximately 50%) of its effect via protein kinase C (PKC) activation. We now show that in addition to ET-1 and 4beta-phorbol-12,13-dibutyrate (PDBu), pervanadate also stimulated PLD activity. Stimulation by pervanadate was not affected by the PKC inhibitor Ro-31-8220 but was abolished by protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin-47. Genistein partially reduced (52%) ET-1 stimulation, which was further attenuated (96%) by Ro-31-8220, indicating that PTKs may account for the PKC-independent arm of ET-1-stimulated PLD activity. Cell-permeable ceramides reduced ( approximately 50%) the activation of PLD by ET-1 and PDBu but not that by pervanadate. Inhibition was also achieved by sphingomyelinase but not with sphingosine. Inhibition by genistein and D-erythro-N-hexanoyl-sphingosine was additive, whereas inhibition by Ro-31-8220 and D-erythro-N-hexanoyl-sphingosine was not, indicating that ceramide affected the PKC-dependent process involved in PLD activation by ET-1. Forskolin, as well as dibutyryl-cAMP and iloprost, attenuated (approximately 50%) the activation of PLD by ET-1 and pervanadate but not that by PDBu. Inhibition by forskolin was prevented by H-89, an inhibitor of protein kinase A. Inhibition by forskolin and ceramide was additive, whereas inhibition by genistein and forskolin was not, indicating that the cAMP/protein kinase A cascade affected the PTK-dependent process involved in PLD activation by ET-1. The data illustrate a cross-talk between separate signaling pathways, resulting in positive and negative regulation of PLD in rat myometrium.     

B cell antigen receptor engagement inhibits stromal cell-derived factor (SDF)-1alpha chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4

The entry of B lymphocytes into secondary lymphoid organs is a critical step in the development of an immune response, providing a site for repertoire shaping, antigen-induced activation and selection. These events are controlled by signals generated through the B cell antigen receptor (BCR) and are associated with changes in the migration properties of B cells in response to chemokine gradients. The chemokine stromal cell-derived factor (SDF)-1alpha is thought to be one of the driving forces during those processes, as it is produced inside secondary lymphoid organs and induces B lymphocyte migration that arrests upon BCR engagement. The signaling pathway that mediates this arrest was genetically dissected using B cells deficient in specific BCR-coupled signaling components. BCR-induced inhibition of SDF-1alpha chemotaxis was dependent on Syk, BLNK, Btk, and phospholipase C (Plc)gamma2 but independent of Ca2+ mobilization, suggesting that the target of BCR stimulation was a protein kinase C (PKC)-dependent substrate. This target was identified as the SDF-1alpha receptor, CXCR4, which undergoes PKC- dependent internalization upon BCR stimulation. Mutation of the internalization motif SSXXIL in the COOH terminus of CXCR4 resulted in B cells that constitutively expressed this receptor upon BCR engagement. These studies suggest that one pathway by which BCR stimulation results in inhibition of SDF-1alpha migration is through PKC-dependent downregulation of CXCR4.     

1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.

The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroid's effects on colonocyte calcium transport and proliferation.     

银杏内酯B对缺血缺氧性脑损伤新生大鼠脑神经元凋亡及P-AKT（ser473）表达的影响

目的 观察银杏内酯B（GB）对缺血缺氧性脑损伤（HIBD）新生大鼠脑皮质神经元凋亡及P-AKT（ser473）蛋白表达的影响。 方法 采用随机数字表法将90只SPF级新生7日龄SD大鼠分为假手术组、缺血缺氧（HI）组及GB组,每组30只。采用经典Rice法建立HIBD动物模型,其中GB组分别于术后0h、24h按每千克体重10mg经腹腔注射GB。各组均于术后不同时间点（包括术后6h、12h、24h及48h）随机处死6只大鼠,采用荧光定量PCR技术检测凋亡关键执行分子Caspase-3 mRNA表达水平,发现GB抑制凋亡最显著的时间点为缺血缺氧后24h;然后增加大鼠并纳入GB+LY294002组,于制模后给予GB及PI3K-AKT通路抑制剂LY294002预干预,进一步探讨缺血缺氧后24h时PI3K-AKT通路在GB抗凋亡中的作用。采用HE染色法观察各组大鼠脑皮质结构变化,采用TUNEL法检测脑皮质神经元凋亡情况,采用免疫组化法检测P-AKT（ser473）蛋白表达情况。 结果 HI组和GB组Caspase-3 mRNA表达于缺血缺氧后6h开始增高,于缺血缺氧后12h进一步上升,于缺血缺氧后24h时达到峰值,随后开始下降;与假手术组比较,HI组和GB组Caspase-3 mRNA表达于缺血缺氧后6h、12h、24h、48h均明显增高,另外GB组Caspase-3 mRNA表达均明显低于HI组,组间差异具有统计学意义（P<0.05）。与假手术组比较,缺血缺氧后24h时HI组、GB组及GB+LY294002组脑皮质神经元Caspase-3表达增强、凋亡阳性细胞增多、P-AKT(ser473)表达减弱,其中HI组和GB+LY294002组Caspase-3表达及凋亡细胞数量均明显超过GB组,P-AKT(ser473)蛋白表达则明显弱于GB组,组间差异均具有统计学意义（P<0.01）。 结论 银杏内酯B具有明显抗神经元凋亡作用,且以缺血缺氧后24h时对神经细胞凋亡的抑制作用最强;此外PI3K-AKT信号通路在GB抗缺血缺氧诱导脑神经元凋亡中发挥重要作用。     

铅与幼鼠脑组织蛋白激酶 C活性及记忆功能改变的关系

背景铅暴露引起的动物学习记忆障碍与脑细胞中蛋白激酶 C(protein kinase C,PKC)活性改变是否有关系呢? 目的研究慢性铅暴露下发育期小鼠大脑组织 PKC活性变化规律及其对小鼠记忆发育的影响. 设计以实验动物为研究对象,随机对照的验证性实验研究. 单位卫生部细胞生物学重点实验室. 材料实验在中国医科大学生物化学与分子生物学实验室完成.选择五六周龄昆明种小白鼠 72只. 方法以不同浓度的醋酸铅饲喂交配后的雌鼠,其幼崽通过哺乳和直接饮水接触铅.在幼崽生后 1 d( P1), 8 d( P8), 15 d( P15), 22 d( P22), 30 d( P30)将其分别处死,取出脑组织.采用 [γ 32P]ATP放射性磷标记法于体外测定铅暴露小鼠大脑组织的 PKC的活性;另外取暴露于不同浓度醋酸铅的发育期小鼠,通过被动回避反应实验进行记忆行为学训练和测试,观察不同浓度的铅对小鼠记忆的影响. 主要观察指标①不同浓度铅暴露对发育期小鼠大脑组织内 PKC活性的影响.②不同浓度铅暴露对发育期小鼠记忆行为的影响. 结果对幼鼠脑 PKC的活性测定显示在发育初期铅暴露小鼠脑中 PKC活性高于正常,发育后期则低于正常,高浓度铅对 PKC活性抑制较强;记忆行为学测试可见低浓度铅使小鼠发育初期记忆曲线升高,而使中长期记忆曲线减低;铅浓度升高,记忆曲线降低. 结论铅对小鼠大脑组织中 PKC活性发育有抑制作用,铅浓度越高,抑制越明显;低浓度铅在短时间内似有刺激记忆的作用,长时间低浓度铅暴露使记忆抑制;高浓度铅使记忆能力降低作用更明显.铅对发育期小鼠脑中 PKC活性及记忆功能的影响有一定的相关性.     

Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, stimulate c-Fos, c-Jun, and c-Myc mRNA expression, mitogen-activated protein kinase activation, and growth in rat aortic smooth muscle cells.

Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroperoxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth.