Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases

Summary Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na⁺/H⁺ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca²⁺, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca²⁺, and an active phorbol ester. These experiments indicate that Na⁺/H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Hydrogen sulphide increases pulmonary veins and atrial arrhythmogenesis with activation of protein kinase C

Hydrogen sulphide (H₂S), one of the most common toxic air pollutants, is an important aetiology of atrial fibrillation (AF). Pulmonary veins (PVs) and left atrium (LA) are the most important AF trigger and substrate. We investigated whether H₂S may modulate the arrhythmogenesis of PVs and atria. Conventional microelectrodes and whole‐cell patch clamp were performed in rabbit PV, sinoatrial node (SAN) or atrial cardiomyocytes before and after the perfusion of NaHS with or without chelerythrine (a selective PKC inhibitor), rottlerin (a specific PKC δ inhibitor) or KB‐R7943 (a NCX inhibitor). NaHS reduced spontaneous beating rates, but increased the occurrences of delayed afterdepolarizations and burst firing in PVs and SANs. NaHS (100 μmol/L) increased I_KATP and I_NCX in PV and LA cardiomyocytes, which were attenuated by chelerythrine (3 μmol/L). Chelerythrine, rottlerin (10 μmol/L) or KB‐R7943 (10 μmol/L) attenuated the arrhythmogenic effects of NaHS on PVs or SANs. NaHS shortened the action potential duration in LA, but not in right atrium or in the presence of chelerythrine. NaHS increased PKC activity, but did not translocate PKC isoforms α, ε to membrane in LA. In conclusion, through protein kinase C signalling, H₂S increases PV and atrial arrhythmogenesis, which may contribute to air pollution‐induced AF.     

Novel phosphorylation site markers of protein kinase C delta activation

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.     

Differential effects of polyamines on rat thyroid protein kinase activities

Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic-AMP-dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6-2.4-fold increase in cytosolic cyclic-AMP-independent (messenger-independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger-independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger-independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid-dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin-regulated ornithine decarboxylase activity in thyroid.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini

Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

Protein kinase C regulates the activity and stability of serotonin N-acetyltransferase

Effects of protein kinase C on protein stability and activity of rat AANAT were investigated in vitro and in vivo. When COS-7 cells transfected with AANAT cDNA were treated with phorbol 12-myristate 13-acetate (PMA), both the activity and protein level of AANAT were increased. These effects of PMA were blocked by GF109203X, a specific inhibitor of PKC. Moreover, PMA increased the phosphorylation of AANAT and induced the formation of AANAT/14-3-3zeta complex. PMA did not affect the basal level of cAMP and did not involve the potentiation of the cAMP production by forskolin, indicating that PKC-dependent activation of adenylyl cyclase was excluded in transfected COS-7 cells. To identify which amino acids were phosphorylated by PKC, several conserved Thr and Ser residues in AANAT were targeted for site-directed mutagenesis. Mutations of Thr29 and Ser203 prevented the increase of enzymatic activity and protein level mediated by PMA. To explore the nature of AANAT phosphorylation, purified rat AANAT was subjected to in vitro PKC kinase assay. PKC directly phosphorylated the rat recombinant AANAT. The phosphopeptides identified by mass spectrometric analysis, and western blotting indicated that Thr29 was one of target sites for PKC. To confirm the effects of the physiological activation of PKC, rat pineal glands were treated with alpha(1)-adrenergic specific agonist phenylephrine. Phenylephrine caused the phosphorylation of endogenous AANAT whereas GF109203X or prazosin, an alpha(1)-adrenergic-specific antagonist, markedly inhibited it. These results suggest that AANAT was phosphorylated at Thr29 by PKC activation through the alpha(1)-adrenergic receptor in rat pineal glands, and that its phosphorylation might contribute to the stability and the activity of AANAT.     

蛋白激酶A和蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用

目的 :研究蛋白激酶A和蛋白激酶C对豚鼠心室肌细胞延迟整流钾电流 (Ik)的影响。方法 :采用电极内液浓度差扩散法进行细胞内给药 ,利用全细胞膜片箝技术测定单细胞Ik。结果 :cAMP15 0 μmol/L使Ik及Ik ,tail(pA/pF)从 13.7± 2 .1和 6 .1± 0 .3增至 18.5± 3.3和 6 .4± 2 .1(P <0 .0 1,n =6 ) ;8 CPT cAMP15 0 μmol/L使电流 (pA/pF)从 11.4± 1.8及 5 .3± 0 .6增至 17.9± 4 .0和 6 .2± 1.3,PKA的选择性抑制剂 6 2 2 1.0 μmol/L的可逆转二者的作用。cAMP使Ik的激活曲线左移 ,半激活电压 (V1/ 2 )从 2 3.3mV移至 18.7mV ,激活曲线斜率 (k)在用药前后变化较小。 10 μmol/LPMA可以分别使Ik和Ik ,tial(pA/pF)从 12 .9± 1.8和 5 .0± 1.7升至 2 3.7± 2 .8和 7.5±1.1。PMA使I V曲线幅值增加 ,并随去极化电压的升高其作用加强 ,同时PMA使通道的激活曲线k从 15 .3mV升到 2 5 .6mV ,但对V1/ 2 基本无影响。结论 :蛋白激酶A和蛋白激酶C均可增加豚鼠心肌细胞Ik,但二者作用特点有所不同     

Effect of protein kinase C on endoplasmic reticulum cholesterol.

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis.     

蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成

采用Spprague-Dawley大鼠胸主动脉中膜、外膜和培养的血管平滑肌细胞(VSMCs)作材料,鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源,闻明蛋白激酶C(PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPG)和细胞因子(TNF-α,IL-1β)处理后,以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育,H7能明显减少LPS、TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成,但HAl004对VSMCs的NO生成无抑制作用,提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、TNF-α和IL-1β引发VSMCs释放NO,同时伴iNOS蛋白表达下调,而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示,PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工;而PTK则以上调iNOS表达而促增NO生成。     

Activators of protein kinase C and 5-azacytidine induce IL-2 receptor expression on human T lymphocytes

Resting human T lymphocytes do not express receptors for interleukin-2, but expression is rapidly induced by exposure to PHA. After maximal expression 2-3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL-2 receptor expression. Moreover, 5-azacytidine, which inhibits cytosine methyltransferase, and hydroxyurea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL-2 receptor expression can be altered in vitro, and that IL-2 receptor number, in combination with IL-2 secretion, may contribute to the regulation of IL-2-dependent immune responses.     

Allosteric network of cAMP-dependent protein kinase revealed by mutation of Tyr204 in the P+1 loop

Previous studies on the catalytic subunit of cAMP-dependent protein kinase (PKA) identified a conserved interaction pair comprised of Tyr204 from the P+1 loop and Glu230 at the end of the alphaF-helix. Single-point mutations of Tyr204 to Ala (Y204A) and Glu230 to Gln (E230Q) both resulted in alterations in enzymatic kinetics. To understand further the molecular basis for the altered kinetics and the structural role of each residue, we analyzed the Y204A and the E230Q mutants using hydrogen/deuterium (H/D) exchange coupled with mass spectrometry and other biophysical techniques. The fact that the mutants exhibit distinct molecular properties, supports previous hypotheses that these two residues, although in the same interaction node, contribute to the same enzymatic functions through different molecular pathways. The Tyr204 mutation appears to affect the dynamic properties, while the Glu230 mutation affects the surface electrostatic profile of the enzyme. Furthermore, H/D exchange analysis defines the dynamic allosteric range of Tyr204 to include the catalytic loop and three additional distant surface regions, which exhibit increased deuterium exchange in the Y204A but not the E230Q mutant. Interestingly, these are the exact regions that previously showed decreased deuterium exchange upon binding of the RIalpha regulatory subunit of PKA. We propose that these sites, coupled with the P+1 loop through Tyr204, represent one of the major allosteric networks in the kinase. This coupling provides a coordinated response for substrate binding and enzyme catalysis. H/D exchange analysis also further defines the stable core of the catalytic subunit to include the alphaE, alphaF and alphaH-helix. All these observations lead to an interesting new way to view the structural architecture and allosteric conformational regulation of the protein kinase molecule.     

G蛋白,蛋白激酶C和Na^＋—H^＋交换在内皮素—1诱导培养心肌细胞肥大反应中的

内皮系-1（ET-1）是一种强的生长因子,并诱导心肌细胞肥大反应。在本实验中,我们探讨了G蛋白、蛋白激酶C（PKC）和Na＋－H＋交换在ET-1诱导的培养新生大鼠心肌细胞肥大反应中的作用。ET-1（10－10～10－7mol／L）促进3H－亮氨酸掺入,增加细胞蛋白质的含量和心肌细胞的表面积,且呈剂量依赖性,它们的EC50分别为5．2×10-10,5．2×10－10和7．3×10－10mol／L。用蛋白激酶C（PKC）抑制剂,Staurosporin（2nmol／L）预处理心肌细胞,可完全阻断ET-1诱导的心肌细胞的这些肥大反应,而蛋白激酶C激动剂,佛波酸酯（PMA）（10－8～10－6mol／L）呈剂量依赖性促进心肌细胞的肥大反应。用Na＋－H＋交换抑制剂,氨氯毗咪（10-4mol／L）预处理心肌细胞,可抑制ET－1诱导的心肌细胞肥大反应,但不影响PMA诱导的心肌细胞肥大反应。百日咳毒素（150ng／ml）预处理心肌细胞,可明显抑制ET－1诱导的心肌细胞肥大反应。这些结果提示,ET-1诱导的培养新生大鼠心肌细胞肥大反应是与百日咳毒素敏感的G蛋白相耦联,蛋白激酶C和Na＋．H＋交换可能在ET-1诱导的心肌细胞肥大反应中是重要的细胞内信使转导途径。