肺间质病患者肺泡巨噬细胞蛋白激酶C活性的变化

目的 探讨肺间质纤维化时蛋白激酶Ｃ（ＰＫＣ）活性的变化。方法 应用放射生测定法检测９名健康者和１５例特发性肺间质纤维化９ＩＰＦ）患者支气管肺泡灌洗液（ＢＡＬＦ）中肺泡巨噬细胞（ＡＭ）的ＰＫＣ活性。结果 ＩＰＦ患者ＢＡＬＦ中ＡＭ总ＰＫＣ活性、南和胸膜ＰＫＣ活性均明显高于健康者（Ｐ〈０．０１和Ｐ〈０．０５）,ＡＭ的总ＰＫＣ活性与ＢＡＬＦ中细胞总数呈正相关（ｒ＝０．５９１７,Ｐ〈０．０５）。结论 ＰＫＣ     

Erythrocytic protein kinase C activity in primary hypertension

Ek P, Toomik R, Eriksson S, Frithz G, Ronquist G, Engström L (University of Uppsala and University Hospital, Uppsala; and Central Hospital, Eskilstuna, Sweden). Erythrocytic protein kinase C activity in primary hypertension. J Intern Med 1998; 243 : 299–305.

Objectives

Increased protein kinase C activity has been reported in erythrocytes from patients with primary hypertension and also from hypertensive rats. In this phenomenological study, we investigated whether a possible increased activity was the result of an augmented amount of enzyme molecules or a more active enzyme.

Design

Collect blood samples, separate erythrocytes from other blood cells. After partial purification of protein kinase C in the erythrocyte lysate, assay the enzyme activity under optimal conditions using a specific peptide substrate.

Setting

Central Hospital in Eskilstuna and University Hospital in Uppsala, Sweden.

Subjects

Healthy individuals: 47 persons (20 women and 27 men). Ten patients with untreated primary hypertension.

Main outcome measures

Erythrocytes were separated from leucocytes and platelets by passing through a cellulose column followed by repeated washings. Some proteins in the erythrocyte lysate interfering with protein kinase C assay were removed by chromatography on DEAE-cellulose.

Results

The mean protein kinase C activity in erythrocytes from healthy individuals was 0.18 ± 0.02 pmol [³²P]phosphate min^?1× 10⁶ cells, regardless of sex and age. The corresponding value for patients with primary hypertension was 0.16 ± 0.04 pmol [³²P]phosphate min^?1× 10⁶ cells.

Conclusions

The amount of protein kinase C, measured as the activity at optimal assay conditions, in erythrocytes from patients with primary hypertension is not critical for the development of moderate hypertension.

     

Melatonin stimulates calmodulin phosphorylation by protein kinase C

Calmodulin (CaM)-dependent processes can be modulated by the availability of Ca(+2), the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post-translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF-7 cells, and selectively activates protein kinase C alpha (PKC alpha) in neuronal N1E-115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC alpha and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [(32)P]-orthoposhospate, and CaM separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC alpha with an EC(50) of 10(-8) m in the presence of the phorbol ester, phorbol-12-myristate-13-acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC alpha in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.     

Increased 19 kDa protein phosphorylation and protein kinase C activity in pressure-overload cardiac hypertrophy

Summary The aim of the study was to determine the role of protein kinase C (PKC) in protein phosphorylation in hypertrophied C. myocytes, particularly the phosphorylation of the 19 kDa protein which corresponds to myosin light chains. In myocardial hypertrophy the PKC activity in the cytosolic fraction of tissue homogenate was increased up to 253 % of control hearts, and in membrane fraction up to 140 % of the control value. Phorbol ester (TPA), the specific activator of protein kinase C, stimulated phosphorylation of the 19 kDa protein obtained from isolated myocytes to 181 ± 9 % of control value in normal and to 248 ± 66 % in hypertrophic myocytes. Taken together, these data suggest that protein kinase C might be involved in the increased phosphorylation of cardiac myosin light chain protein in myocardial hypertrophy.     

Characterization of apoptosis induced by protein kinase C inhibitors and its modulation by the caspase pathway in acute promyelocytic leukaemia

Acute promyelocytic leukaemia (APL;M3) is a unique form of acute myelogenous leukaemia characterized by t(15;17) translocation. The induction of apoptosis via inhibiting protein kinase C (PKC) has been recently viewed as a promising tool for the eradication of several malignant disorders. In the present study, we investigated the effect of two different protein kinase C inhibitors, Gö6976 and safingol, on the induction of apoptosis in the APL cell line NB4 and its all trans retinoic acid (ATRA)‐resistant variant NB4.306. The effect of the PKC inhibitors on leukaemic cells obtained from three APL patients was also studied. We also evaluated the possible involvement of the caspases in apoptosis induced by PKC inhibitors. Significant time‐ and concentration‐dependent apoptotic changes were demonstrated using Gö6976 and safingol. In addition, our results demonstrated that the caspases were involved in the apoptosis induced by the PKC inhibitors. In conclusion, our study illustrates that the PKC inhibitors Gö6976 and safingol induce apoptosis in APL and hence could be potential therapeutic agents for the treatment of this disease.     

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP₂) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP₂-hydrolyzing PLC activity (PIP₂-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP₂-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca²⁺-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP₂-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP₂-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.Until April 31, 1990 N. Makita, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-Ku Sapporo 060, Japan After May 1, 1990 N. Makita, Division of Nephrology, S-3223, Medical Center North, Vanderbilt University, Nashville. Tennessee 37232, USA     

Inhibition of calmodulin and protein kinase C by amiodarone and other class III antiarrhythmic agents

Summary Class III antiarrhythmic agents may prolong refractoriness via modulation of ion channels, which may be sensitive to Ca²⁺ regulatory proteins or enzymes. Accordingly, the purpose of this study was to quantitate the effects of several structurally diverse class III antiarrhythmic agents on calmodulin-regulated enzymes and protein kinase C activity, and to evaluate the ability of these agents and known calmodulin antagonists to prolong cardiac refractoriness in vivo. The rank order of potency (IC₅₀;M) of selected class III antiarrhythmic agents and reference calmodulin antagonists as inhibitors of calmodulin-regulated phosphodiesterase activity were: calmidazolium (0.12 M)>amiodarone (0.62 M)>desethylamiodarone (1.5 M)>trifluoperazine (4.3 M), bepridil (5 M)>W-7 (7.5 M), clofilium (13 M). Similar concentration-related inhibition was evident in a second calmodulin-regulated system, inhibition of myosin light-chain phosphorylation and superprecipitation light-chain phosphorylation and superprecipitation of arterial actomyosin. Sotalol and tetraethylammonium were inactive at 100 M. Protein kinase C activity was also inhibited by some of these agents; desethylamiodarone (IC₅₀=11 M) was more potent than the reference agent, H-7 (IC₅₀=79 M), or amiodarone (38% inhibition at 100 M) and clofilium (32% inhibition at 100 M). In vivo, the minimally effective doses required to increase ventricular effective refractory doses required to increase ventricular effective refractory periods in paced guinea pigs were (in mg/kg) bepridil, sotalol [1]>clofilium [3]>amiodarone [10]>W-7, desethylamiodarone [20]. No changes in refractory period were noted with maximum testable doses of calmidazolium or trifluoperazine. These studies show that some, but not all, class III antiarrhythmic agents are effective and potent calmodulin antagonists or protein kinase C inhibitors. Moreover, some calmodulin antagonists are effective at prolonging refractoriness in vivo. However, a lack of correlation between these agents suggests that these mechanisms are not solely responsible for the prolongation of refractoriness of all class III agents.     

Melatonin increases stress fibers and focal adhesions in MDCK cells: participation of Rho-associated kinase and protein kinase C

Melatonin cyclically modifies water transport measured as dome formation in MDCK cells. An optimal increase in water transport, concomitant with elevated stress fiber (SF) formation, occurs at nocturnal plasma melatonin concentrations (1 nm) after 6 hr of incubation. Blockage in melatonin-elicited dome formation was observed with protein kinase C (PKC) inhibitors. Despite, this information on the precise mechanism by which melatonin increases SF formation involved in water transport is not known. Focal adhesion contacts (FAC) are cytoskeletal structures, which participate in MDCK membrane polarization. SF organization and vinculin phosphorylation are involved in FAC assembly and both processes are mediated by PKC, an enzyme stimulated by melatonin; in these processes also involved is Rho-associated kinase (ROCK). Thus, we studied FAC formation and the ROCK/PKC pathway as the mechanism by which melatonin increases SF formation and water transport. The results showed that 1 nM melatonin and the PKC agonist phorbol-12-miristate-13-acetate increased FAC. The PKC inhibitor GF109203x, and the ROCK inhibitor Y27632, blocked increased FAC caused by melatonin. ROCK and PKC activities, vinculin phosphorylation and FAC formation were increased with melatonin. The PKC inhibitor, GF109203x, abolished both melatonin stimulated FAC in whole cells and ROCK activity, indicating that ROCK is a downstream kinase in the melatonin-stimulated PKC pathway in MDCK cultured cells that causes an increase in SF and FAC formation. Data also document that melatonin modulates water transport through modifications of the cytoskeletal structure.     

Further identification of protein kinase C isozymes in mouse epidermis

In the current study, the protein kinase C (PKC) isozymes present in mouse epidermis have been identified using immunological and chromatographic methods. Six PKC isozymes, PKC, PKC, PKC, PKC, PKC, and PKC, were identified in unfractionated epidermal preparations by protein immunoblotting. The subcellular distribution and presence of these isozymes was further verified by hydroxyapatite (HA) chromatography with the exception of PKE, which could not be detected following HA chromatography. The five PKC isozymes recovered following HA chromatography were detected in both epidermal cytosol and particulate fractions, although PKC was found in a much higher proportion relative to the other PKC isozymes in the particulate fraction using histone H1 as the substrate. The biochemical properties of the epidermal PKC isozymes partially purified by HA chromatography agreed with those reported for other tissues and further supported their immunological identification in epidermal preparations. The activities of HA chromatography peaks corresponding to PKC, PKC, and PKC were found to be dependent on both Ca²⁺ and phosphatidylserine (PtdSer), whereas, the activities of HA peaks corresponding to PKC and PKC were Ca²⁺-independent but PtdSer-dependent. The HA peak corresponding to PKC also displayed a characteristic biphasic modulation by arachidonic acid (activation at low, inactivation at high concentrations) and inactivation by preincubation with PtdSer. PKC activity was also characteristic, in that it was dependent on PtdSer and was not increased by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Some differences in substrate specificity were also observed between the epidermal PKC isozymes. The presence of multiple isozymes of PKC in mouse epidermis suggests that the different isozymes may play distinct roles in signal transduction and tumor promotion in this tissue.Abbreviations PKC protein kinase C - HA hydroxyapatite - PtdSer phosphatidylserine - TPA 12-O-tetradecanoylphorbol 13-acetate This work was supported by USPHS grants CA 38871 (J.D.), CA 57596 (J.D.) and core grant CA 16672     

大鼠脑出血后蛋白激酶C同工酶和细胞内钙离子与细胞凋亡的关系

目的 探讨大鼠脑出血后不同时间点血肿周围神经细胞蛋白激酶C（PKC）同工酶（nPKCδ和cPKCγ）、细胞内游离钙浓度与细胞凋亡之间的关系及其意义.方法 将36只成年健康Wistar大鼠,随机分为正常对照组和脑出血后0.5、6、24、72、120h组,每组6只大鼠.采用胶原酶法制备大鼠脑出血模型,利用免疫组化观察PKC同工酶的表达,Fura-2/AM荧光探针法检测细胞内游离钙浓度,TUNEL法检测细胞凋亡.结果 脑出血后血肿周围nPKCδ、cPKCγ表达及细胞内游离钙变化与细胞凋亡数呈正相关,各指标大致为0.5h开始增加,6～24h达高峰,72h开始下降,120 h明显下降（r分别为0.970、0.984,P均＜0.01）.结论 脑出血后血肿周围nPKCδ、cPKCγ表达上调和细胞内钙超载与神经细胞凋亡关系密切,提示细胞信号转导途径异常可能参与了脑出血后神经细胞损伤的病理生理学过程.     

Alterations in protein kinase C system of colonic epithelium during fasting-refeeding

In the present study, we compared (1) incorporation of [³H]dThd into DNA, (2) total protein kinase C (PKC) activity, (3) the subcellular distribution of PKC, and (4) PKC isozyme (, and ) mass in colonic mucosal scrapings and isolated superficial and proliferative colonic epithelial cells from 48-hr fasted, 48-hr fasted-refed, and adlibitum-fed rats. Total colonic mucosal PKC activity and PKC mass were higher and thein vivo rate of [³H]dThd incorporation into mucosal DNA was markedly depressed in 48-hr fasted rats compared toad libitum-fed or fasted-refed rats. These alterations were localized predominantly to the proliferative pool of colonic epithelial cells. Despite an 11-fold increase in mucosal DNA synthesis, no alterations in total mucosal PKC activity were detected in fasted-refed rats compared to rats fedad libitum. Moreover, no differences in the subcellular distribution of PKC were noted among any of the dietary groups. Intrarectal instillation of deoxycholate activated PKC and increased DNA synthesis 1.5-to 2-fold. Deoxycholate-induced increases in DNA synthesis, but not those induced by refeeding, were inhibited by treatment of rats with the PKC inhibitors H-7, sphingosine, or staurospaurine. The results do not support a role for PKC in the mediation of increased proliferative activity of colonic mucosa induced by refeeding.     

蛋白激酶C的空间不均一性分布与室性心律失常发生的关系

目的 探讨蛋白激酶C(PKC)不均一性表达与室性心律失常(VA)之间的联系.方法 30只兔经Iangendorff心脏灌流系统,分别在基线、PKC激动剂(phorbol-12-myristate-13-acetate,100 nmol/L)及PKC抑制剂(bisindolylmaleimide,500 nmol/L)状态下,应用程控刺激(S1S2)及动态S1刺激记录心室10个局部位点的动作电位时限(APD)、有效不应期(ERP)以及交替和VA发生阈值,分析APD及ERP的空间离散度(COv).同时运用Real-time PCR技术检测心室各点PKC亚型的mRNA表达量.结果 应用PKC激动剂可导致各记录位点APD及ERP发生不均一性缩短,使离散度指标COV-APD90和COV-ERP显著增大(P＜0.05),左心室较明显(COV-APD90:0.05±0.01比0.08±0.03,P＜0.01;COV-ERP:0.05±0.01比0.09±0.03,P＜0.01).在PKC激动剂作用下,VA诱发率较基线状态下显著升高(86.7％比40.0％,P＜0.05),并且各点发生APD交替现象和VA的阈值明显降低(P＜0.05).但PKC抑制剂可减小复极离散度以及升高交替和VA阈值,并使之恢复至基线水平.分析各点PKC亚型表达发现PKC-α和PKC-β的mRNA表达存在空间差异性,且该特征与PKC激动剂灌注前后APD及ERP差值的标准化比值呈现相似分布趋势.结论 PKC激活导致心室空间电生理特征的不均一性主要归因于PKC亚型的差异性分布,为VA的发生提供了潜在条件.     

Inhibition of granulosa cell differentiation by dioctanoylglycerol--a novel activator of protein kinase C

The actions of dioctanoylglycerol, a novel and specific activator of protein kinase C, on granulosa cell steroidogenesis and LH/hCG receptor induction were examined in vitro. Granulosa cells were cultured for 2 days in media containing 100 nM androstenedione. FSH treatment stimulated progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-P) and estrogen production by 60- to 80-fold over basal levels. Treatment with dioctanoylglycerol increased progesterone production 3-fold over basal levels but failed to affect 20 alpha-OH-P or estrogen production. Concomitant treatment of cells with FSH and increasing amounts of dioctanoylglycerol produced dose-dependent inhibition of FSH-stimulated progesterone, 20 alpha-OH-P and estrogen production with IC50 values of 75, 40 and 50 microM, respectively. Dioctanoylglycerol also inhibited production of all three steroids induced by forskolin, an activator of adenylate cyclase, and dibutyryl cAMP. The effects of dioctanoylglycerol on cAMP responses to FSH were also examined. The increase in both intracellular and extracellular cAMP at 1 h following treatment with FSH was suppressed in the presence of dioctanoylglycerol as was the extracellular accumulation of cAMP at 48 h of culture. FSH induction of LH/hCG receptors, known to be a cAMP-mediated event, was also inhibited in the presence of dioctanoylglycerol. In contrast, a thio-derivative of dioctanoylglycerol (3-thio-1,2-dioctanoylglycerol), which does not activate protein kinase C, failed to inhibit both steroidogenesis and LH/hCG receptor induction in response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diacylglycerol/protein kinase C signalling: a mechanism for insulin resistance?

Abstract. It is proposed that an intracellular cycle exists to limit or terminate the insulin signal. The cycle involves increased synthesis of sn-1,2-diacylglycerol (DAG) in response to insulin. The DAG activates protein kinase C (PKC) which phosphorylates glycogen synthase either directly or through other protein kinases to render it inactive. Protein kinase C may also inhibit the insulin receptor by phosphorylation of receptor serine residues. Insulin resistance could then arise as a consequence of a persistent increase in DAG levels. Such an increase could occur in three different ways. Chronic hyperinsulinaemia could increase DAG levels by de-novo synthesis from phosphatidic acid, by hydrolysis of phosphatidylcholine, or by hydrolysis of glycosyl-phosphatidylinositol; DAG is also formed by hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP₂). This reaction, known as the ‘PI response,’ may be the connection between hypertension and insulin resistance. A third mechanism for an increase in DAG involves neural abnormalities. Thus, muscle denervation in the rat is characterized both by a profound insulin resistance and a large increase in DAG. It is possible that a similar increase occurs in humans and may explain the association between denervation, inactivity, and insulin resistance.     

抗磷脂抗体与抗活化蛋白C抵抗关系的研究

目的探讨抗磷脂抗体(APLs)与抗活化蛋白C(APCR)的关系。方法APCR检测以受检血浆加入活化蛋白C(APC)后的活化的部分凝血活酶时间(APTT)与未加APC血浆的APTT的敏感比值(APC-SR)表示,当APC-SR<2.0为APCR阳性。抗心磷脂抗体(ACL)检测和抗β2糖蛋白(β2GP)-Ⅰ抗体检测采用酶联免疫吸附试验。狼疮抗凝物(LA)采用激活的APTT测定法。结果LA与APCR有显著相关性(!2=16.332,P=0.008);而ACL-IgG、ACL-IgM和ACL-IgA3种ACL亚型均与APCR无统计学关系(!2=0.674,P=0.412;!2=2.978,P=0.071;!2=3.546,P=0.06);抗β2GP-Ⅰ抗体与APCR之间有显著相关性(!2=6.179,P=0.012)。APCR阳性组患者与阴性组患者的血栓形成及反复流产发生率差异有统计学意义(!2=7.347,P=0.007)。同时含有两种以上抗磷脂抗体17例(42.5%),APCR发生率达70.6%;血栓和/或反复流产64.7%,与含有一种APLs的病例比较差异有统计学意义(P=0.002)。结论LA和抗β2GP-Ⅰ抗体与APCR密切相关,APLs对蛋白C系统有抑制作用,APCR可能是诱发血栓的危险因素之一。APLs抗体的联合检测对并发血栓可能有预测价值。     

蛋白激酶C与钙敏感受体在心肌缺血预适应中的保护作用

目的 探讨蛋白激酶C(PKC)与钙敏感受体(CaR),在心肌缺血预适应(IPC)中的保护作用.方法 采用细胞培养方法 ,体外培养大鼠乳鼠心肌细胞,模拟缺血预适应模型,实验分为7组:①正常对照组(C),②缺血再灌注组(1/R),③IPC组,④IPC+PKC抑制剂组(IPC+PKCI),⑤IPC+PKCI+CaR激动剂组(IPC+PKCI+CARS),⑥IPC+CaRS组,⑦IPC+CaR抑制剂组(IPC+CaRI).分别用TUNEL,Hoechst 33342染色法检测细胞凋亡,四甲基偶氮唑比色法(MTT)观察细胞存活率,Western Blot法检测胞浆内caspase-12,CaR,钙蛋白酶(calpain)表达.结果 光镜下,I/R组细胞核缩小,呈强蓝色荧光,染色质浓缩,出现凋亡小体,其他各实验组有不同程度的荧光增强,尤以IPC+PKCI+CaRS组多见强蓝色荧光细胞核.心肌细胞存活率和凋亡率,I/R组[(62.99±0.65)%,(19.13±0.87)%],IPC组[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI组[(71.09±0.52)%.(20.46±0.81)%],IPC+PKCI+CaRS组[(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS组[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI组[(85.81±0.60)%,(13.12±0.69)%]明显低于或高于对照组[(100.00)%,(6.02±0.31)%],除IPC+CaRI组心肌细胞存活率外,其他各组与对照组比较差异有统计学意义(P＜0.05或＜0.01).Western Blot测定,胞浆白CaR蛋白在IPC+PKCI,IPC+CaRS,IPC+PKCI+CaRS组表达较IPC组高,IPC+CaRI组较IPC组低,caspase-12蛋白在I/R,IPC+CaRS,IPC+PKCI+CaRS 组,相对分子质量(胁)为60×103的活性片段表达均较高,calpain在I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRS组表达均高于对照组和IPC+CaRI组,其中I/R组最高,对照组最低,IPC+CaRI组次之.结论 PKC与CaR受体之间的相互作用在IPC中可以通过减少肌浆网钙释放而起到对心肌的保护作用.Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning DU Li-juan,WANG Yah-li,SUN Zhi-rui,ZHAO Ya-jun,LI Quan-feng,WANG Li-na,ZHANG Wei-hua Departmem of Pothophysioloty,Harbin Medical University,Harbin 150081,China     

Role of myosin light-chain kinase and protein kinase C in pepsinogen secretion from guinea pig gastric chief cells in monolayer culture

We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C (PKC) in pepsinogen secretion from guinea pig gastric chief cells using a monolayer culture system of chief cells and an enzyme immunoassay system for guinea pig pepsinogen. An MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), significantly inhibited both the basal pepsinogen secretion and the secretion by carbamylcholine chloride (carbachol) or ionomycin without affecting intracellular free Ca²⁺ concentration ([Ca²⁺]i), but not by 12-O-tetradecanoylphorbol-13-acetate (TPA) or forskolin. A PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), significantly reduced the pepsinogen secretion by carbachol or TPA, but not by forskolin or ionomycin, and did not affect the basal secretion and the [Ca²⁺]i elevated by carbachol or ionomycin. We concluded that: (1) MLCK plays an important role in basal and drug-stimulated pepsinogen secretion, (2) MLCK is involved in the Ca²⁺-dependent intracellular pathway but not in the cyclic adenosine monophosphate (cAMP) dependent pathway, (3) PKC is irrelevant to activation of MLCK, and (4) increases in cAMP and [Ca²⁺]i are independent of activation of PKC.This study was supported by Grant (03670371) from the Ministry of Education, Japan.