Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have established that the terminally differentiated ventricular cardiac muscle cell of the adult rat reinitiates semiconservative DNA replication when grown in culture (W. C. Claycomb and H. D. Bradshaw, Jr., 1983, Dev. Biol. 90, 331-337). Work reported here shows that several growth factors and chemicals will stimulate this DNA synthetic activity in a concentration-dependent manner. Autoradiographic experiments establish that this stimulated DNA synthesis is due to cells not previously synthesizing DNA being induced to enter the S phase of the cell cycle. By far the greatest stimulation (250%) is observed with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Fifty ng/ml is the optimal concentration, and the maximal effect is observed 5 days after adding TPA. TPA also substantially increases the protein content of the cultured myocytes. Diacylyglcerols (DAG) induce these same changes, indicating that the effect of TPA is mediated by protein kinase C. The morphology of the cultured cardiac muscle cells is profoundly altered by TPA and DAG. TPA- and DAG-treated myocytes spread more thinly on the surface of the culture flask, acquire multiple nuclei, and undergo nucleolar fragmentation. The myofibrillar ultrastructure of the treated cells becomes almost totally disorganized, and intermediate filaments and rough endoplasmic reticulum accumulate in the cytoplasm. These TPA results suggest a possible relationship between the degree of ultrastructural differentiation of the ventricular cardiac muscle cell and DNA synthetic activity. This easily altered cellular plasticity should be very useful for studies of the regulation of cardiac muscle cell proliferation and cell differentiation.
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PMID:Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells. 337 63

Calcium-mobilizing agonists act by stimulating the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) to inositol 1,4,5-trisphosphate and diacylglycerol (DG). In response to such agonists cells also produce inositol 1,3,4-trisphosphate but this isomer is unlikely to influence calcium mobilization. Application of inositol 1,4,5-trisphosphate (Ins1,4,5P3) to permeabilized cells results in a rapid release of calcium from the endoplasmic reticulum. Structure-activity studies reveal that the vicinal phosphates on the 4- and 5-positions are essential for releasing calcium whereas the phosphate on the opposite side enhances the affinity of Ins1,4,5P3 for its putative receptor. The flow of calcium across the endoplasmic reticulum appears to be electrogenic and requires an opposite flow of potassium to neutralize charge movements. Diacylglycerol, acting through protein kinase C, does not play a direct role in calcium signalling but it does modulate various aspects of the InsP3/Ca2+ pathway. The DG/protein kinase C pathway can influence both the formation and hydrolysis of PtdIns4,5P2 and can alter the responsiveness of various processes to the action of calcium. The Ins1,4,5P3/Ca2+ signal pathway functions throughout the life history of cells to regulate such diverse activities as egg maturation and fertilization, growth, secretion, metabolism, neural activity, and perhaps excitation-contraction coupling in skeletal muscle.
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PMID:Inositol trisphosphate and calcium mobilization. 349 45

Rat liver homogenate or cell fractions deacylate 12-O-tetradecanoyl phorbol 13-acetate (TPA) in vitro mainly by conversion to phorbol 13-acetate. The highest specific activity is located in the microsomal fraction. The deacylation is inhibited by bis-(4-nitrophenyl) phosphate, a selective inhibitor of nonspecific carboxylesterases. Only two of five purified esterases from rat liver endoplasmic reticulum deacylate TPA. These two esterases have formerly been characterized as acylcarnitine hydrolases and the more active one is also a potent diacylglycerol lipase. Its TPA-hydrolyzing activity is inhibited by other substrates like 1-naphthylacetate, lauroylcarnitine, or dioleoyl glycerol. The results support the view that phorbol esters act like structural analogs of diacylglycerols, not only with respect to their activating effect on protein kinase C, but also as substrates for the same lipases.
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PMID:The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase. 371 Nov 10

Stimulation of exocytosis in exocrine glands is associated with an increased phosphorylation of several particulate proteins. Irrespective of the type of secretagogue (cAMP-dependent agonists, calcium-dependent agonists, calcium ionophores, phorbol esters) exocytosis is always accompanied by an enhanced phosphorylation of the ribosomal protein S6. It is shown by an analysis of the phosphopeptide pattern of the in vivo and the in vitro phosphorylated S6 protein that the protein kinase responsible for phosphorylation of the S6 protein during enhanced exocytosis is protein kinase C. This is so irrespective of whether the agonist uses cAMP or calcium as second messenger. Experiments with isolated guinea pig parotid gland lobules reveal that not only the acetylcholine analog carbamoylcholine, but also the beta-agonist isoproterenol lead within seconds to an increased formation of diacylglycerol. As diacylglycerol increases the affinity of protein kinase C for calcium this finding would explain why the phosphorylation pattern of the S6 protein reflects activation of protein kinase C also under conditions where (as in the case of stimulation with beta-agonists) cAMP is the primary second messenger. It would further explain why the changes of the phosphorylation of individual histones observed during agonist-induced exocytosis in the parotid gland are quite similar for isoproterenol on one hand and carbamoylcholine on the other. A 22 K protein which becomes phosphorylated only when cAMP serves as second messenger is located in the membrane of the endoplasmic reticulum. A possible relationship of this protein with the calcium transport ATPase of the endoplasmic reticulum is under investigation.
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PMID:Regulation of protein kinases in exocrine secretory cells during agonist-induced exocytosis. 407 96

Histamine release from mast cells is intimately related with degranulation. When basic histamine releasers such as compound 48/80 were applied extracellularly to isolated rat mast cells by means of microelectrophoresis, localized degranulation was evoked near the tip of micropipet in a few seconds. In response to the second electrophoretic application at the opposite side of the membrane of the same mast cells, similar local degranulation was induced. This fact clearly indicates that local degranulation does not damage mast cells to the extent of blocking following degranulation. As intracellular electrophoretic application of compound 48/80 caused a swelling of mast cell, although no degranulation was elicited. When antigen-antibody reaction was induced in a single rat mesentery mast cell by means of microelectrophoresis, the application of antigen was made extracellularly or intracellularly. At the site of extracellular application, localized degranulation and histamine release were evoked. Histamine release was evidenced by the disappearance of histamine fluorescence in the degranulated area. Neither degranulation nor histamine release was induced by intracellular application of antigen. In freeze-fracture electronmicroscopy of the resting rat mast cells, intra-membrane particles (IMPs) were randomly distributed on the plasma membrane. When sensitized cells were exposed to antigen, IMPs were markedly dispersed so as to surround bulging regions of the membrane elicited by swollen granules. As the particles gathered at the periphery of the bulges, actually no particle was seen on the protuberant region. When rat mast cells loaded with quin 2 were exposed compound 48/80 in a Ca-free medium, a marked increase of quin 2 fluorescence was noticed, indicating that Ca2+ was released from intracellular Ca store. The binding of 45Ca was at its peak in the fractions where the highest activity of glucose-6-phosphatase, a marker enzyme for the endoplasmic reticulum, when organelles of mast cells were fractionated. This may indicate that intracellular Ca store is endoplasmic reticulum. It has been shown that microfilaments, and microtubules play some important roles in histamine release from rat mast cells. When permeabilized mast cells were stimulated with Ca2+, a translocation of protein kinase C from cytosol to membrane fraction was observed. This leads to phosphorylation of vimentin, one of intermediate filaments. In membrane skeletons of rat mast cells, alpha- and beta-fodrin, ankyrin and actin were found by means of western blotting analysis. It was supposed that membrane skeleton may be useful as a barrier between the plasma membrane and the granule membrane.
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PMID:[Development of the research in the field of histamine release]. 751 63

The export of vesicular stomatitis virus glycoprotein (VSV-G) from the endoplasmic reticulum (ER) involves sorting and concentration, and has been proposed to require the function of heterotrimeric G proteins. To begin to identify the basic elements of a potential signaling pathway involved in vesicle assembly, we have examined whether protein kinase C (PKC) is required for ER to Golgi transport. Calphostin C, a specific inhibitor of the highly conserved cysteine-rich C6H2 motif present in the regulatory domain of PKC was found to be a potent inhibitor of export of VSV-G and vesicle budding from the ER in vivo and in vitro (IC50 approximately 60 nM). In contrast, the diacylglycerol analog phorbol 12-myristate 13-acetate, which activates PKC, enhanced the migration of VSV-G from the ER to pre-Golgi intermediates. Neither reagent had detectable effects on the oligomerization of VSV-G prior to export nor perturbed transport of protein between compartments of the Golgi stack. In contrast to the striking effects of calphostin C, reagents that inhibit the function of the catalytic domain of PKC (including the general kinase inhibitor staurosporine, as well as the more specific inhibitors H-7, H-8, pseudosubstrate inhibitor, or chelerythrine) did not inhibit export from the ER. Export was also insensitive to down-regulation of various PKC isoforms. These results suggest that a novel protein containing the conserved C6H2 motif may serve as a potential link in a signaling pathway regulating vesicle budding from the ER.
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PMID:Export of protein from the endoplasmic reticulum is regulated by a diacylglycerol/phorbol ester binding protein. 752 13

The expression and distribution of prolactin (PRL) mRNA and their alterations induced by estrogen and bromocriptine were investigated using non-radioisotopic in situ hybridization (ISH) at the electron microscopic (EM) level. Our EM-ISH studies using biotinylated oligonucleotide probes showed that estrogen induced whirling changes of the rough endoplasmic reticulum (RER) of female rat PRL cells and increased transcription of PRL genes located on the polysomes of the whirling RER. The presence of mammosomatotroph cells in the rat pituitary gland was also verified in our EM-ISH studies. After bromocriptine administration, PRL cells contained many secretory granules due to the inhibition of secretion. Pre- and post-embedding EM-ISH and northern hybridization studies revealed that bromocriptine induced the distorted, vesiculated, and dilated RER, and also the suppressed PRL mRNA expression. The activity of protein kinase C (PKC), which mediates PRL gene expression, tended to be elevated by estrogen and suppressed by bromocriptine. Therefore, it is considered that the ultrastructural and quantitative changes in PRL mRNA expression evoked by estrogen and bromocriptine may be mediated by the intracellular signal transduction system, including PKC.
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PMID:Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes. 758 58

Intracellular Ca2+ (Cai2+) stores contribute significantly to Ca2+ signaling in many types of cells. We studied the role of inositol trisphosphate (InsP3)-sensitive Ca2+ stores, a principal Cai2+ store that presumably is within the endoplasmic reticulum (ER), in cell signaling by examining the effect of thapsigargin (Tg), an ER Ca2+ pump inhibitor that depletes the ER Ca2+ pool, on ACTH secretion. Preincubation for 6-24 h with 2-20 nM Tg had no effect on the resting cytosolic free Ca2+ concentrations ([Cai2+]) but inhibited the ionomycin-stimulated spike-type increase in [Cai2+], which is mediated by InsP3-independent Cai2+ release from the ER, in a dose-dependent (IC50, 4 nM) and time-dependent manner. In ER Cai(2+)-depleted cells, the spike phase (initial 5 min) of the ACTH secretory response to arginine vasopressin (AVP), which is mediated by InsP3-induced Cai2+ release, was also attenuated (IC50, 7.3 nM). However, the spike phase of the ACTH secretory response to AVP was inhibited to a much greater degree than the spike-type response to ionomycin, suggesting that ER Cai2+ stores might have functions other than simply providing Ca2+ for InsP3-stimulated Cai2+ release. Tg pretreatment (IC50, 12 nM) also markedly inhibited the sustained plateau (final 15-min) phase of the ACTH secretory response to AVP, which is mediated by diacylglycerol-induced activation of protein kinase C and subsequent influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels (VSCC), but had no effect on the sustained (full 20 min) response to dioctanoylglycerol that directly activates protein kinase C. Tg had no effect on specific cell binding of [125I]AVP or on specific cell binding of [3H]phorbol 12,13-dibutyrate (except at 20 nM Tg), an index of protein kinase C concentration, or on protein kinase C activity. AVP significantly stimulated inositol trisphosphate accumulation, but pretreatment with Tg completely abolished this effect of AVP, whereas [3H]myoinositol incorporation into membrane-associated inositol lipids and inositol phosphates was unaffected. Thus, Tg-induced depletion of ER Cai2+ stores inhibited both the spike and plateau phases of the ACTH secretory response to AVP, presumably by inhibiting phospholipase C activity and the resulting generation of InsP3 and diacylglycerol. Preincubation with Tg inhibited, in a dose-dependent (IC50, 13 nM) and time-dependent manner, the sustained ACTH secretory response to corticotropin-releasing hormone (CRH) that is mediated by cAMP-induced activation of protein kinase A and Cae2+ influx via L-type VSCC, and the sustained response to forskolin, which directly activates adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of inositol trisphosphate-sensitive calcium stores in the regulation of adrenocorticotropin secretion by perifused rat anterior pituitary cells. 758 88

Hormones that elevate cytosolic Ca2+ concentrations ([Ca2+]cyt) often use Ca2+ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca2+ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca2+]cyt by vasopressin or other agonists causes Ca2+ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) prior to permeabilization with digitonin. Mitochondrial Ca2+ accumulation was determined by following the ionomycin-induced Ca2+ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenyl-arsenoxide and rotenone. The results indicate that agents that elevate [Ca2+]cyt cause a significant Ca2+ accumulation in the mitochondria. Excessive Ca2+ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca2+ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca2+ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca2+ accumulation following elevation of elevation of [Ca2+]cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.
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PMID:Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition. 759 32

Glioma C6 cells treated with 12-0-tetradecanoyl-phorbol-13-acetate, TPA (10 nM and 100 nM) manifested slow increase in intracellular calcium concentration ([Ca2+]i), dependent upon both Ca2+ release from intracellular stores and Ca2+ entry, and ranging from 50 to 500 nM in different cells. The effect of TPA was abolished by the down-regulation procedure and by protein kinase C inhibitors, such as staurosporine (100 nM), suramin (100 microM), and sphingosine (100 microM), pointing to a role of protein kinase C (PKC) in this process. On the other hand, thapsigargin (100 nM), a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, produced a rapid increase in [Ca2+]i (up to 800 nM). This increase consisted of a transient initial phase followed by sustained elevation in [Ca2+]i, typical of Ca2+ release from intracellular stores and of Ca2+ entry, respectively. However, when the cells were exposed to TPA (100 nM) prior to thapsigargin (100 nM), then thapsigargin produced only a transient rise in [Ca2+]i. We suggest that TPA, a PKC activator, affects thapsigargin-induced Ca2+ entry, probably by PKC-mediated changes in cytoskeleton structures.
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PMID:Changes in Ca2+ concentration in phorbol ester and thapsigargin treated glioma C6 cells. The role of protein kinase C in regulation of Ca2+ entry. 762 33


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