Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The contractile responses of cultured rat and calf endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts (FB) to vasoactive mediators (thrombin, serotonin, bradykinin, and histamine), forskolin, and cytochalasin B were compared. Cells were grown on a pliable silicone membrane, and contraction was assessed, using time-lapse video microscopy, by recording changes in the wrinkling of the silicone as the cells exerted tension on the surface. We found that all cells contracted in the presence of serum or thrombin and that VSMC and FB also contracted with serotonin stimulation. Bradykinin and histamine were not contractants in this system. Discrepancies between these results and reports of changes in permeability of endothelial layers in vitro and in vivo may be due to (1) the vascular segment from which EC were studied or (2) the possibility that certain mediators may provoke a noncontractile response that results in gap formation. Thus changes in vascular permeability, which occur during inflammation, may have both contractile and noncontractile components. Forskolin, known to indirectly inhibit myosin light-chain kinase activity, and cytochalasin B were potent relaxants, suggesting a similar smooth muscle-like contractile mechanism for all three cell types.
 ...
PMID:Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts. 158 60

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.
 ...
PMID:Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells. 171 Sep 33

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.
 ...
PMID:Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets. 241 Feb 92

1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.
 ...
PMID:Stimulated calcium efflux from fura-2-loaded human platelets. 245 43

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin. We now show that another agent of possible importance in mediating proliferation of cells adjacent to the endothelial cell layer, transforming growth factor-beta (TGF-beta), also induced c-sis expression in these cells. In addition, we have studied the effect of agents that increase intracellular cAMP levels upon the induction of endothelial cell c-sis mRNA. The adrenergic agonists isoproterenol and norepinephrine blocked the elevation of cellular c-sis mRNA accompanying exposure to either thrombin or TGF-beta. This effect was mediated through beta-adrenergic receptors, since propranolol but not phentolamine reversed the inhibition. Forskolin, a direct activator of adenylate cyclase, also blocked induction of c-sis mRNA by thrombin and TGF-beta and inhibited the release of PDGF activity into the media of these cells. Basal, as well as stimulated c-sis mRNA levels were attenuated by these agents that increase cellular cAMP levels. These data suggest that increased cAMP production inhibits the expression of c-sis encoded mitogens by endothelial cells, and that c-sis expression is subject to bidirectional regulation in these cells.
 ...
PMID:Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta. 304 Jul 21

The biochemical mechanisms responsible for regulating cellular platelet-derived growth factor expression are incompletely understood. Our previous studies have shown that platelet-derived growth factor B/c-sis mRNA levels are induced in human renal microvascular endothelial cells by either thrombin or transforming growth factor (TGF-beta), while exposure to agents which elevate cAMP levels blocks the induction responses. The current studies use combined transcription run-off and message decay rate experiments to show greater than 3-fold increases in rate of transcription after stimulation with either thrombin or TGF-beta. c-sis message has a 70-90-min half-life under basal conditions that is effectively unaltered by thrombin or TGF-beta. Forskolin does not decrease the stability of c-sis mRNA, although it attenuates transcription increases seen with inducing agents. TGF-beta induction of c-sis transcription is mediated independent of the protein kinase C (Ca2+- and phospholipid-dependent enzyme)-mediated responses to phorbol ester, as it remains intact following down-regulation of protein kinase C response; TGF-beta and phorbol elicit additive induction. Inhibitory effects of cAMP upon transcription act distal to early thrombin-receptor-coupled increases in phosphatidylinositol turnover and are capable of turning off TGF-beta-activated transcription after activation has been established. Both inducing and suppressing agents alter endothelial platelet-derived growth factor B/c-sis mRNA expression dominantly through effects upon rates of transcription, cAMP suppression of transcription is dominant, and TGF-beta and phorbol esters mediate induction of transcription through distinct pathways.
 ...
PMID:Distinct pathways mediate transcriptional regulation of platelet-derived growth factor B/c-sis expression. 319 52

Washed platelets in the absence of physiological activators possess gravity inducible shape change, which is paralleled by an increase of phosphatidic acid (PA), polyphosphoinositides (PPI) and an inhibition of PGE1 and Gpp(NH)p- stimulated adenylate cyclase (AC) activity. Incubation of platelets at 37 degrees C for 1 hr decreases (32P)PPI and restores their response to low doses of thrombin (0.015 U/ml). Simultaneously an increase of PGE1- and Gpp(NH)p- stimulation of AC is observed. The relaxation of the platelets influences predominantly the cAMP levels without significantly affecting the dissociation constants of the stimulators. Forskolin-induced activation of AC is the same in stimulated and relaxed platelets. It is suggested that the initial increase of PA inhibits the coupling of regulatory and receptor proteins to AC and has no effect on the catalytic unit.
 ...
PMID:Inhibition of adenylate cyclase activity during the reversible shape change in human platelets. 336 46

The effects of diterpene forskolin on the human platelet release reaction and on platelet protein phosphorylation were studied. This drug is shown to have the same effects as other agents which increase cAMP levels, namely, it inhibits the secretory response to diverse agonists and causes changes in the phosphorylation of several specific proteins. An increase of the 32P content is seen in the MW 47 000, 24 000 and 21 000 polypeptides while a decrease is observed in the MW 41 000 and 27 000 and 20 000 species. Forskolin also inhibits the changes in protein phosphorylation pattern induced by the powerful platelet secretagogue, thrombin. Our results relate the effects of antagonists of platelet secretion such as prostaglandins more closely to changes in cAMP levels and in protein phosphorylation than to other possible effects of the receptor-ligand interaction, which is by-passed by the use of forskolin. Our results also provide additional evidence involving these changes in the mechanisms which regulate the secretory process in platelets.
 ...
PMID:Effects of diterpene forskolin on the release reaction and protein phosphorylation of human platelets. 608 7

Endothelial cells are central in fibrinolysis because of their high production of both activators (t-PA, uPA) and inhibitors (PAI-1). The t-PA and PAI-1 synthesis could be regulated by signals transduction at several cellular levels. The purpose of this in vitro study, on cultured endothelial cells, was to explore the receptor/second messenger regulation of the t-PA and PAI-1 synthesis. Quiescent confluent human umbilical vein endothelial cells, cultured in passage 1, were exposed to different test substances. Samples from the conditioned medium were collected after 16 and 24 h and analysed for t-PA and PAI-1 antigen. All data presented were related to the data from control dishes (= 100%), in the same experiment. The results from the present study (mean +/- 95% confidence interval) demonstrated the following. (1) Forskolin, with a documented direct cAMP-inducing effect, decreased the basal PAI-1 production to 61 +/- 15%, and Na-nitroprusside, with a documented cGMP-inducing effect, increased the basal PAI-1 production to 141 +/- 38% without affecting the basal t-PA production. The surface receptor agonists isoprenalin or ephedrine, which indirectly affect adenylate cyclase, had no effect on t-PA or PAI-1 production. (2) Phorbolester (PMA), which directly activates proteinkinase C (PKC), increased the basal t-PA and PAI-1 production to 350 +/- 71%, and 163 +/- 35% respectively. (3) Thrombin, but not endothelin-1 (ET-1), increased the basal t-PA and PAI-1 production to 195 +/- 34% and 136 +/- 18%, respectively, indicating an PKC-mediated thrombin effect.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Complex intracellular signal transduction regulates tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) synthesis in cultured human umbilical vein endothelium. 756 35

Direct measurement of nitric oxide (NO) release is pivotal for understanding its role in the regulation of vascular tone. However, data on the direct measurement of NO have been scarce. Recent description of NO-selective electrode has prompted us to examine NO release from endothelial cells using this approach. In the present study, we continuously monitored [NO] in the incubation medium conditioned by cultured human umbilical vein endothelial cells (HUVEC) with an amperometric NO-sensor. The HUVEC released NO on stimulation with several agonists such as alpha-thrombin, bradykinin, L-arginine and ionomycin; the responses were characterized by an initial rise and a subsequent sustained increase. Activation of Ca/calmodulin system resulted in a robust elevation in [NO], occasionally displaying an oscillatory component. Calmidazolium pretreatment attenuated the ionomycin-induced response. Pretreatment with phorbol ester suppressed the ionomycin-induced NO release from HUVEC. Forskolin pretreatment did not modify NO release elicited by ionomycin. These findings indicate that the synthesis/release of NO in endothelial cells is a Ca/calmodulin dependent step. Activation of protein kinase C interferes with the Ca/calmodulin-induced activation of NOS in endothelial cells. Thus, the present study shows that NO synthase is a substrate for phosphorylation by different kinases which modulate the activity of the enzyme as determined by continuous monitoring of NO release from endothelial cells using a specific NO-sensor.
 ...
PMID:Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells. 851 71


1 2 Next >>