Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.     

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.     

Thrombin receptor peptide inhibits thrombin-induced increase in endothelial permeability by receptor desensitization

Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent manner. The peak increase in [Ca2+]i in response to 100 microM TRP-14 or 0.1 microM alpha-thrombin was similar (i.e., 931 +/- 74 nM and 1032 +/- 80 nM, respectively), which was followed by a slow decrease with t1/2 values of 0.73 and 0.61 min, respectively. Extracellular Ca2+ chelation with 5 mM EGTA abolished the sustained increases in [Ca2+]i induced by either TRP-14 or alpha-thrombin. alpha- thrombin (0.1 microM) increased transendothelial [125I]albumin permeability, whereas TRP-14 (1-100 microM) had no effect. Coincubation of 100 microM TRP-14 with 1 microM DIP-alpha-thrombin also did not increase permeability over control values. Stimulation of BPAEC with 0.1 microM alpha-thrombin induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane indicative of PKC activation, whereas TRP-14 had no effect at any concentration. TRP-14 at 100 microM desensitized BPAEC to thrombin-induced increases in [Ca2+]i and transendothelial permeability. The Ca2+ desensitization was reversed after approximately 60 min, and this recovery paralleled the recovery of the permeability response. These findings indicate that the TRP-14-induced Ca2+ mobilization in the absence of PKC activation is insufficient to increase endothelial permeability. In contrast, the increase in endothelial permeability after alpha-thrombin occurred in conjunction with Ca2+ mobilization as well as PKC activation. TRP-14 pretreatment prevented the alpha-thrombin-induced increase in endothelial permeability secondary to desensitization of the Ca2+ signal. The results suggest that combined cytosolic Ca2+ mobilization mediated by TRP-14 and PKC activation mediated by a TRP-14-independent pathway are dual signals responsible for the thrombin-induced increase in vascular endothelial permeability.     

Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple Galpha(i) and Galpha(q) proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Galpha(i), Galpha(q), and Galpha(12/13) proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Galpha(i), Galpha(q), and Galpha(12/13) signaling pathways, and a protein kinase C (PKC)-alpha inhibitor, G?-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.     

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells.

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.     

Thrombin-stimulated increases in cytosolic Ca2+ level and gonadotropin-releasing hormone release in GT1-7 neurons.

The effects of thrombin on cytosolic calcium levels ([Ca2+]cyt), and on gonadotropin-releasing hormone (GnRH) release, were characterized in cultured GT1-7 neurons. GnRH release from GT1-7 neurons was pulsatile with an average pulse amplitude of 14.3+/-5.8 pg x min x ml(-1) and an average pulse duration of 21.3+/-4.2 min. The [Ca2+]cyt response to 0.005 to 0.2 U/ml thrombin was saturable and concentration dependent (EC50 = 0.0268 U/ml). Ethyleneglycotetraacetic acid (EGTA) chelation of extracellular Ca2+ resulted in an approximately 70% attenuation of thrombin-stimulated increase in [Ca2+]cyt. By use of a special superfusion system, a 5-min exposure to 0.1 U/ml thrombin significantly increased the amplitude (193.2+/-67.8 pg x min x ml(-1); P = 0.001) but not the duration (22.5+/-2.4 min; P = 0.8) of GnRH release. These results suggest that thrombin increases [Ca2+]cyt and GnRH release from GT1-7 neurons via specific membrane-bound receptors.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol.

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.     

Different signaling pathways involved in glucose- and cell swelling-induced insulin secretion by rat pancreatic islets in vitro.

BACKGROUND: The objective was to compare signal transduction pathways exploited by glucose and cell swelling in stimulating insulin secretion. METHODS: Isolated rat (Wistar) pancreatic islets were stimulated in vitro by 20 mmol/l glucose or 30% hypotonic medium (202 mOsm/kg) in various experimental conditions. RESULTS: Glucose did not stimulate insulin release in calcium free medium. Cell swelling-induced insulin release in calcium free medium, even in the presence of the membrane permeable calcium chelator BAPTA/AM (10 micromol/l). Protein kinase C (PKC) inhibitor bisindolylmaleimide VIII (1 micromol/l) abolished the stimulation of insulin secretion by glucose but did not affect the swelling-induced insulin release. PKC activator phorbol 12-13-dibutyrate (1 micromol/l) stimulated insulin secretion in medium containing Ca2+ and did not potentiate insulin secretion stimulated by hypotonic extracellular fluid. Dilution of the medium (10-30%) had an additive effect on the glucose-induced insulin secretion. Noradrenaline (1 micromol/l) abolished glucose-induced insulin secretion but did not inhibit hypotonic stimulation either in presence or absence of Ca2+. CONCLUSION: Glucose- and swelling-induce insulin secretion through separate signal transduction pathways. Hyposmotic stimulation is independent from both the extracellular and intracellular Ca2+, does not involve PKC activation, and could not be inhibited by noradrenaline. These data indicate a novel signaling pathway for stimulation of insulin secretion exploited by cell swelling.     

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs.

Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.     

Signal transduction of a tissue interaction during embryonic heart development.

During early cardiac development, progenitors of the valves and septa of the heart are formed by an epithelial-mesenchymal cell transformation of endothelial cells of the atrioventricular (AV) canal. We have previously shown that this event is due to an interaction between the endothelium and products of the myocardium found within the extracellular matrix. The present study examines signal transduction mechanisms governing this differentiation of AV canal endothelium. Activators of protein kinase C (PKC), phorbol myristate acetate (PMA) and mezerein, both produced an incomplete phenotypic transformation of endothelial cells in an in vitro bioassay for transformation. On the other hand, inhibitors of PKC (H-7 and staurosporine) and tyrosine kinase (genistein) blocked cellular transformation in response to the native myocardium or a myocardially-conditioned medium. Intracellular free calcium concentration ([Ca2+]i) was measured in single endothelial cells by microscopic digital analysis of fura 2 fluorescence. Addition of a myocardial conditioned medium containing the transforming stimulus produced a specific increase in [Ca2+]i in "competent" AV canal, but not ventricular, endothelial cells. Epithelial-mesenchymal cell transformation was inhibited by pertussis toxin but not cholera toxin. These data lead to the hypothesis that signal transduction of this tissue interaction is mediated by a G protein and one or more kinase activities. In response to receptor activation, competent AV canal endothelial cells demonstrate an increase in [Ca2+]i. Together, the data provide direct evidence for a regional and temporal regulation of signal transduction processes which mediate a specific extracellular matrix-mediated tissue interaction in the embryo.     

Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.

Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.     

Lack of inhibition of thrombin-induced rise in intracellular Ca2+ levels and 5-hydroxytryptamine secretion by 1-oleoyl-2-acetylglycerol in human platelets

The effect of 1-oleoyl-2-acetylglycerol (OAG) on the thrombin-induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5-hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10-50 micrograms/ml) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10-50 micrograms/ml) 10 s to 5 min before or 10-60 s after addition of threshold concentrations of thrombin (less than 0.03 U/ml) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin-induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin-induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.     

大鼠星形胶质细胞组织因子活性的表达及其调控

本实验观察了基础培养条件和凝血酶刺激条件下,大鼠星形胶质细胞组织因子活性的表达及其信号传递途径。结果显示,基础条件下,Ａ２３１８７（４ｂｒｏｍｏｃａｌｃｉｕｍｉｏｎｏｐｈｏｒｅ）和佛波醇脂（ｐｈｏｒｂｏｌ１２ｍｙｒｉｓｔａｔｅ１３ａｃｅｔａｔｅ,ＰＭＡ）能明显提高星形胶质细胞组织因子活性的表达,而三氟吡啦嗪（ｔｒｉｆｌｕｏｐｅｒａｚｉｎｅ,ＴＦＰ）和１（５异喹啉磺胺）３甲基哌嗪［１（５ｉｓｏｑｕｉｎｏｌｉｎｙｌｓｕｌｆｏｎｙｌ）３ｍｅｔｈｙｌｐｉｐｅｒａｚｉｎｅ,Ｈ７］则降低星形胶质细胞组织因子活性的表达。凝血酶能明显增加星形胶质细胞表达组织因子活性;当凝血酶与ＴＦＰ或Ｈ７联合应用时,凝血酶的刺激作用受到明显抑制。实验表明,星形胶质细胞在基础培养条件下能表达组织因子,凝血酶能刺激它表达组织因子活性。Ｃａ２＋／钙调素和ＰＫＣ途径参与了在基础条件以及凝血酶刺激条件下星形胶质细胞组织因子活性的表达。     

Thrombin receptors activate G(o) proteins in endothelial cells to regulate intracellular calcium and cell shape changes

Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but not G(i1/2) minigenes. We conclude that the G(o) proteins play a role in thrombin signaling distinct from G(i1/2) proteins, which are mediated through their Gbetagamma subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.