Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Na+/H+ antiporter, which regulates intracellular pH in virtually all cells, is one of the best examples of a mitogen- and oncogene-activated membrane target whose activity rapidly changes on stimulation. The activating mechanism is unknown. A Na+/H+ antiporter complementary DNA fragment was expressed in Escherichia coli as a beta-galactosidase fusion protein, and a specific antibody to the fusion protein was prepared. Use of this antibody revealed that the Na+/H+ antiporter is a 110-kilodalton glycoprotein that is phosphorylated in growing cells. Mitogenic activation of resting hamster fibroblasts and A431 human epidermoid cells with epidermal growth factor, thrombin, phorbol esters, or serum, stimulated phosphorylation of the Na+/H+ antiporter with a time course similar to that of the rise in intracellular pH.
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PMID:Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. 215 36

Growth of Chinese hamster lung fibroblasts (CCL39) on thrombin as sole mitogen is dependent on phosphatidylinositol (PI) metabolism and activation of the Na+/H+ antiporter. By modifying a H+ suicide selection developed for the isolation of antiporter mutants in these cells, we enriched for and isolated CCL39 variants deficient in the thrombin mitogenic response (thrombin nongrowers). These mutants retain alternate mitogenic mechanisms and, hence, grow well on media containing serum. When challenged with thrombin, the mutants show decreased, increased, or unchanged levels of inositol phosphates produced as compared with wild type cells. One of the mutants (D1-6b) has decreased inositol phosphates production not only with thrombin but also with serotonin (5-hydroxytryptamine) and AlF4-, suggesting a defect distal to the thrombin receptors. Extracts of this mutant reveal marked decreased phospholipase C activity toward PI. From the different phenotypes of the thrombin nongrowers, it is clear that the selection is general and that mutants with various biochemical defects should lead to a better understanding of the PI cycle as well as of functions essential to mitogenesis.
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PMID:Hamster fibroblasts defective in thrombin-induced mitogenesis. A selection for mutants in phosphatidylinositol metabolism and other functions. 276 25

Stimulation of platelets with thrombin, ADP and epinephrine has recently been shown to activate a Na+/H+ antiporter, with a resulting alkalinization of the cytoplasm. Unlike thrombin, however, epinephrine is incapable of directly activating phospholipase C, but is well known to potentiate the effects of thrombin on this enzyme and other subsequent steps of platelet activation. Therefore, we have studied the involvement of the Na+/H+ antiporter in this aspect of epinephrine action to see whether alkalinization of platelet cytosol could be a requirement for agonists to stimulate inositol phospholipid hydrolysis and mobilize cellular Ca2+ stores. Alpha-thrombin induced the rapid formation of inositol trisphosphate with a parallel mobilization of intracellular Ca2+ stores. Epinephrine alone had no effect on either of these parameters. The response to thrombin desensitized over a period of minutes, and after this had occurred, epinephrine was able to activate phospholipase C and induce the release of intracellular Ca2+. This showed that epinephrine was able to recouple thrombin receptors to phospholipase C, and this appeared to be mediated by the same mechanism which is involved in potentiation by epinephrine of thrombin-stimulation of phospholipase C. These effects of epinephrine were not altered by inhibition of the Na+/H+ antiporter with ethylisopropylamiloride or by use of the Na+/H+ ionophore, monensin. We conclude that epinephrine potentiates thrombin-induced responses by a mechanism which is unrelated to its effects on the Na+/H+ antiporter, and this is not a requirement for thrombin-induced phospholipase C activation.
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PMID:The Na+/H+ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine. 283 79

In resting Chinese hamster fibroblasts (CCL39) alpha-thrombin rapidly stimulates several biochemical events implicated in the mitogenic response, including the breakdown of inositol phospholipids, activation of a plasma membrane Na+/H+ antiporter, phosphorylation of ribosomal protein S6 and increased expression of the proto-oncogene c-myc. Complete removal of the growth factor during cellular G0/G1 transit precludes the re-initiation of DNA synthesis. The present study was designed to examine the fate of alpha-thrombin-activated early events following growth factor inactivation. In cells stimulated for 30 min with alpha-thrombin, neutralization of the growth factor results in: (i) immediate arrest of inositol phosphate formation, (ii) rapid inactivation of Na+/H+ exchange, (iii) deactivation of the S6 phosphorylating system and (iv) strong reduction of c-myc mRNA level. Our findings that commitment for DNA synthesis as well as persistent activation of 'early' cellular events requires continual growth factor stimulation suggest that: (i) growth factor-induced transmembrane signals have a short life and (ii) the generation of these signals during the 8 h of the pre-replicative phase is required for G0-arrested cells to enter the S phase.
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PMID:alpha-Thrombin-induced early mitogenic signalling events and G0 to S-phase transition of fibroblasts require continual external stimulation. 299 66

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.
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PMID:Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets. 301 59

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.
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PMID:Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. 302 26

Chinese hamster embryo fibroblast cells (CHEF/18) possess a plasma membrane-associated, amiloride-sensitive Na+/H+ antiporter that affects intracellular pH (pHi) and is activated by growth factor addition. Our results using 14C-benzoic acid distribution indicate that both epidermal growth factor (EGF) and thrombin are capable of causing rapid rises in the pHi of CHEF/18 cells. The maximal shift induced by these factors is 0.20 to 0.25 pH units above the basal unstimulated level. Distinctive differences were observed between the modes of action of these two growth factors. Sequential additions revealed that the rise in pHi due to EGF was additive with that caused by diacylglycerols (DAG), while that of thrombin was not. Furthermore, exposure of cells to the phorbol ester PMA for a prolonged period of time in order to down-regulate protein kinase C (pkC), or treatment with the pkC inhibitor H-7, abolished the pHi response to thrombin but not to EGF. In contrast, incubation of cells in nominally calcium-free medium or with the calmodulin antagonists W-7 or trifluoperazine (TFP) decreased only the ability of EGF to cause changes in pHi. These data suggest that there are two distinct mechanisms for activation of the Na+/H+ antiporter in CHEF/18 fibroblast cells and thus provide an example of the use of alternative modes for the modulation of intracellular processes.
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PMID:Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts. 304 Jul 79

Growth factors (alpha-thrombin and insulin) activate a Na+/H+ antiport in G0/G1-arrested Chinese hamster lung fibroblasts (CCL39). In this report, we have examined the influence of intracellular pH on this exchange activity, measured by initial rates of amiloride-sensitive 22Na+ uptake, in the absence and presence of growth factors. Our results indicate the following. 1) In quiescent as in mitogen-stimulated cells, Na+/H+ antiport is regulated by internal H+ in an allosteric way, whereas, in contrast, interactions with external H+ and Na+ obey simple saturation kinetics. 2) The growth factor-induced activation of Na+/H+ exchange, which, under physiological conditions, is responsible for a sustained cytoplasmic alkalinization, is due to an increased affinity for internal H+ (the apparent pK is shifted by approximately 0.3 pH unit towards alkaline pH values). Therefore, we propose that growth factors promote a conformational change of the Na+/H+ antiporter, possibly at the level of an internal modifier site(s).
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PMID:Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+. 608 24

The interference of several new hexadecylphosphocholine analogues with mitogenic signal transduction was investigated in NIH3T3 fibroblasts by studying the effects of these agents on thrombin-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation and the subsequent Ca2+ release, on protein kinase C (PKC) in cell-free extracts, on the PKC-mediated activation of the Na+/H+ antiporter and on c-fos induction. The compounds investigated include hexadecylphosphocholine (HePC), octadecyl-[2-(N-methyl-piperidinio)-ethyl]-phosphate (D20133), octadecyl-(N,N-dimethyl-piperidinio-4-yl)-phosphate (D21266); octadecyl-[2-(trimethyl-arsonio)-ethyl]-phosphate (D21805) and hexadecylphospho-L-serine (HePS). The data indicate that (i) all compounds inhibit the thrombin-induced progression of growth-arrested NIH3T3 cells into S phase with similar IC50 values; (ii) the common denominator of all compounds is a reduction of Ins(1,4,5)P3 formation, resulting in an attenuation of Ca2+ release; (iii) the direct interaction with PKC does not significantly contribute to the antitumor activity of these agents; (iv) the new HePC congeners D21266, D21133 and D21805 affect the same targets as HePC, i.e. PKC and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC). The lower toxicities of these compounds cannot be explained by a less pronounced inhibition of PKC or PLC, respectively.
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PMID:Interference of new alkylphospholipid analogues with mitogenic signal transduction. 763 30

1. In Fura-2 preloaded human platelets, the increase in cytosolic calcium induced by alpha-thrombin was reduced by some L- and D-arginine ester compounds the IC50 (microM) values of which were 7.4 for TAEE, 56.9 for BAEE, 77.6 for TAME, 560 for T(d)AME, 656.3 for L-ArgOMe and 2206.7 for D-ArgOMe. alpha-tosyl-L-Arginine, L- and D-arginine were inactive. 2. The inhibitory activity of the L-arginine esters was not modified when platelets were pretreated with 100 microM N omega-monomethyl-L-arginine. 3. The L-arginine esters did not increase cyclic GMP content in platelets either in the presence or absence of indomethacin and apyrase at rest and after alpha-thrombin stimulation. 4. The kinetic parameters of platelet Na+/H+ antiporter (amiloride-inhibitable, evaluated after cytosolic nigericin-induced acidification) were modified by L- and D-arginine esters, while the native amino acids were ineffective. 5. The inhibitory effects of the L- and D-arginine esters on platelet activation appear to be mainly due to their inhibitory effect on Na+/H+ antiporter.
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PMID:Effects of L- and D-arginine and some related esters on the cytosolic mechanisms of alpha-thrombin-induced human platelet activation. 822 Aug 81


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