© 1997 American Heart Association, Inc.
Articles |
A Role for Tyrosine Phosphorylation in Generation of Inositol Phosphates and Prostacyclin Production in Endothelial Cells
the Department of Pharmacology, University of Iceland, Reykjavik (A.H., H.H., K.M., G.T.); and the Departments of Medicine (H.H., G.T.) and Clinical Chemistry (M.K.), Landspitalinn, University Hospital, Reykjavik, Iceland.
Correspondence to Gudmundur Thorgeirsson, Department of Pharmacology, University of Iceland, PO Box 8216, 128 Reykjavik, Iceland.
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Abstract |
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We have examined the effects of the protein tyrosine phosphatase inhibitor pervanadate on activation of signal transduction in human umbilical vein endothelial cells. Endothelial cells responded to pervanadate treatment by increasing tyrosine phosphorylation of cellular proteins, including phospholipase C (PLC)
1, generating inositol phosphates (IPs), releasing arachidonic acid, and producing prostacyclin (prostaglandin [PG] I2). The dose and time responses for these events were similar. Tyrosine phosphorylation and formation of IPs in response to pervanadate were reduced by both staurosporine and genistein. Short-term incubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which inhibits thrombin-induced IP generation, did not affect the IP response to pervanadate. To investigate the possible involvement of tyrosine phosphorylation in thrombin or histamine-induced IP generation and PGI2 production, we examined the effects of costimulation with pervanadate and either thrombin or histamine. These responses proved to be different. While the tyrosine phosphorylation of PLC1 was enhanced after cotreatment with thrombin and pervanadate compared with pervanadate alone, costimulation with pervanadate and histamine resulted in no more tyrosine phosphorylation of PLC1 than after pervanadate alone. Similarly, while cotreatment with pervanadate and thrombin caused synergistic increase in IP generation, costimulation with pervanadate and histamine resulted in an additive response. However, PGI2 responses to costimulation of pervanadate with either thrombin or histamine were both synergistic. Furthermore, stimulation with histamine, thrombin, or pervanadate all caused tyrosine phosphorylation of a mitogen-activated protein kinase (ERK1/p44). The results suggest that a tyrosine phosphorylation–dependent mechanism has a role in the phosphoinositide signal transduction pathway of human endothelial cells. Moreover, thrombin- but not histamine-induced generation of IPs appears to be partly caused by tyrosine phosphorylation of PLC1.
Key Words: endothelial cells • tyrosine phosphorylation • inositol phosphates • prostacyclin • PLC1
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Introduction |
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Endothelial cells respond to a variety of receptor-mediated stimuli by releasing arachidonic acid, from which PGI2, a potent vasodilator and inhibitor of platelet aggregation, is produced. Activation of phosphatidylinositol-PLC, which generates the intracellular second messengers inositol trisphosphate and diacylglycerol, is an important and possibly necessary step in agonist-activated PGI2-production.1 2 3 Phosphatidylinositol-PLC comprises a family of several isoforms, which are activated by different receptors and different mechanisms.4 5 Several tyrosine kinase–containing receptors, such as the receptors for EGF and PDGF, activate PLC
in certain cell types by inducing tyrosine phosphorylation.4 Activation of the T-cell receptor, which does not contain an intrinsic PTK domain, has also been shown to induce tyrosine phosphorylation of PLC1 through cytosolic PTKs.6 7 8 In contrast, the ß-isoform (PLCß) is activated by a G-protein–coupled receptor stimulation. Activation of G-proteins by AlF4- leads to generation of IPs and PGI2 production in endothelial cells.9 Recently, Bowden and coworkers10 have shown that the regulation of vascular endothelial cells by ATP acting at the G-protein–coupled P2Y and P2U purinoceptors involves tyrosine phosphorylation and suggests that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production. Also, Fleming and coworkers11 have reported that Ca2+ influx is modulated by tyrosine kinase inhibitors in endothelial cells. However, nothing has been reported about the possible involvement of tyrosine phosphorylation of PLC in these cells. Stimulation of various receptors containing tyrosine kinase domains, including those for basic fibroblast growth factor, EGF, PDGF, insulin, and insulin-like growth factor, has been found to induce proliferation of endothelial cells in vitro.12 13 Activation of these receptors stimulates tyrosine kinase activity and subsequent tyrosine phosphorylation of specific proteins.14
Thrombin and histamine both stimulate inositol phospholipid hydrolysis in endothelial cells.1 2 3 Their receptors couple to G-proteins, suggesting subsequent activation of the ß-isoform of PLC.15 16 However, thrombin has also been shown to induce tyrosine phosphorylation of PLC in platelets.17 18
In the present study we have used pervanadate, a powerful PTPase inhibitor,19 20 21 to enhance the phosphotyrosine content of endothelial cells. We have demonstrated that tyrosine phosphorylation of PLC represents an alternate pathway for activation of human endothelial cells. Furthermore, such activation of PLC serves a selective role, since it is activated by thrombin and not by histamine.
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Methods |
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Materials
[3H]Arachidonic acid and myo-[3H]inositol, ECL reagents, and nitrocellulose membranes were obtained from Amersham International; Morgan's medium 199, fetal calf serum, antibiotics, and genistein were from GIBCO; catalase, BSA (essentially fatty-acid free), HEPES, LiCl, histamine, staurosporine, thrombin, and TPA were from Sigma; the collagenase was either from Sigma or provided by The Science Institute of Iceland; anion-exchange resin was from Bio-Rad; tissue-culture plates were from Nunc; monoclonal antibodies to phosphotyrosine and MAP kinase (ERK1/p44) and anti-mouse IgG-HP were obtained from Affinity; antibodies to PLC
1 were from Affinity, UBI, or Santa Cruz Biotechnology; and anti-rabbit IgG-HP and protein A/G PLUS–agarose were from Santa Cruz Biotechnology.
Preparation of Pervanadate
Pervanadate was prepared as described by Pumiglia and coworkers,22 by mixing 1 part of 500 mmol/L H2O2 with 5 parts of 10 mmol/L sodium orthovanadate in modified Tyrode's solution (145 mmol/L NaCl; 5 mmol/L KCl; 5.5 mmol/L glucose; 0.04 mmol/L CaCl2; 1 mmol/L MgCl2; and 10 mmol/L HEPES, pH 7.4) and incubating at room temperature for 15 minutes before use. In the majority of experiments, 300 U/mL of catalase was added after formation of pervanadate to remove residual H2O2. The pervanadate solution was used within half an hour. The concentration of pervanadate is denoted by the vanadate concentration in the mixture.
Endothelial Cell Culture
Endothelial cells were cultured from human umbilical veins as previously described.1 Briefly, the cells were harvested by collagenase digestion and seeded on 35-mm culture dishes in medium 199 containing 20% fetal calf serum and antibiotics (penicillin, 100 U/mL, and streptomycin, 100 µg/mL). The culture dishes were incubated at 37°C in humidified air with 5% CO2. The medium was changed 24 hours after seeding the cells and every 3 days thereafter until the cells had reached confluence.
Formation of IPs
Confluent cell cultures were incubated for 20 to 36 hours in 1 mL of Morgan's medium 199 containing 20% fetal calf serum, antibiotics, and 3 µCi of myo-[3H]inositol per milliliter. Before the experiments were carried out, the cells were washed twice with medium containing 20 mmol/L LiCl. The experiments were performed in 1 mL of this solution with agonists and/or inhibitors in the indicated concentrations. At the time points indicated in each experiment, the medium was removed for measurement of PGI2, and 1 mL of ice-cold TCA was added to terminate reactions. IPs were separated on columns of anion-exchange resin and quantified by liquid scintillation counting.1 Accumulation of combined IPs (inositol monophosphates, inositol bisphosphates, and inositol trisphosphates) in the presence of LiCl was used as a measure of PLC activity.
Arachidonic Acid Release
Confluent cells were incubated for 24 hours in Morgan's medium 199 containing 20% fetal calf serum, antibiotics, and 1 µCi of [3H]arachidonic acid per milliliter. Before the experiments, the cells were washed twice with medium containing BSA (1 mg/mL) and kept in this solution. Twenty minutes later, a portion of the medium was removed and an equal volume of a solution containing the agonist was added to give the indicated final concentrations. At the time points indicated, another portion of the medium was removed. All portions were then counted in a scintillation counter for quantification of arachidonic acid and its labeled metabolites.
PGI2 Production
To measure PGI2 production of the cells, the medium was subjected to a radioimmunoassay for 6-oxo-PGF1
, a stable catabolite of PGI2, as previously described.1
Electrophoresis and Immunoblotting
Confluent endothelial cells were stimulated for indicated times with different concentrations of pervanadate and/or thrombin or histamine in 1 mL of Morgan's medium 199. The reactions were stopped by aspirating the medium and adding 200 µL of SDS sample buffer. The samples were boiled and centrifuged for 5 minutes before electrophoresis on 7.5% or 10% SDS-polyacrylamide gels according to the method of Laemmli.23 Separated proteins were then transferred to nitrocellulose in a semidry transfer unit (Hoefer) for 90 minutes at 40 mA. After blocking with 1% BSA in wash buffer (0.01 mol/L Tris, pH 7.5; 0.1 mol/L NaCl; and 0.1% Tween 20) overnight at 4°C, the nitrocellulose membranes were probed with antibodies to PLC1, phosphotyrosine, or MAP kinase by incubation in blocking buffer for 1 hour at room temperature. After several washes, the immunocomplexes were detected with horseradish peroxidase conjugated to mouse or rabbit IgG, using the ECL substrate system. Immunoblots that were reprobed with a different antibody were stripped with two sequential incubations, each for 30 minutes at 70°C, in a solution of 2% SDS, 100 mmol/L 2-mercaptoethanol, and 62.5 mmol/L Tris-HCl (pH 6.8), and incubated overnight in blocking buffer before detection as described, with the appropriate antibody.
Immunoprecipitation
After stimulation of cells, reactions were stopped by washing once with ice-cold PBS before lysis for 20 minutes on a rocker with 200 µL of modified RIBA buffer (50 mmol/L Tris-HCL, pH 7.4; 1% NP-40; 0.25% SDS; 150 mmol/L NaCl; 1 mmol/L EGTA; 1 mmol/L PMSF; 1 mmol/L Na3VO4; 1 mmol/L NaF; and 1 µg/mL each of aprotinin, leupeptin, and pepstatin) per dish. Cell lysates were clarified by centrifugation (13 000g for 15 minutes) and precleared with protein A/G PLUS–agarose (30 minutes on ice) before incubation with PLC1 antibodies and rocking the mixture for 2 hours on ice. A 20-µL slurry of protein A/G PLUS–agarose was then added and incubation continued overnight on ice. Immunocomplexes were collected by centrifugation and washed three times with cold PBS, after which pellets were boiled in SDS sample buffer and proteins resolved by electrophoresis in 10% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose before immunoblotting and were detected as above.
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Results |
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Effects of Pervanadate on IP Generation, Arachidonic Acid Release, and PGI2 Production
After exposure of endothelial cells to pervanadate, generation of IPs was stimulated in a dose- and time-dependent manner. As shown in Fig 1
, the maximal level of IPs, almost sixfold that of control, was reached at 20 µmol/L pervanadate. For this experiment, pervanadate was prepared without addition of catalase, resulting in a decline in the response at doses above 20 µmol/L owing to the inhibitory effects of excess H2O2. Such inhibition by H2O2 of IP formation has been demonstrated after stimulation of endothelial cells by thrombin and histamine.24 The dose-response curve for arachidonic acid release was similar to that for IPs (Fig 1). Fig 2, which is from an experiment carried out in the presence of catalase, shows that the dose-response curve for PGI2 production in response to pervanadate is also similar to the one for IPs. Fig 3 illustrates the time course of formation of IPs and PGI2 in [3H]inositol-labeled cells exposed to 20 µmol/L pervanadate. Production of IPs began before the initiation of PGI2 production. Between 10 and 20 minutes, there was a rapid rise in 6-oxo-PGF1, after which it leveled off. In contrast, IP generation continued throughout the 40 minutes of the experiment. Separately, H2O2 (125 µmol/L) or vanadate (12.5 µmol/L) had negligible effects on all the parameters studied (results not shown).
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) or [3H]arachidonic acid (
) were incubated with various concentrations of pervanadate for 20 minutes at 37°C. Data are expressed as radioactivity in IPs (
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), measured by radioimmunoassay. Each point is the mean of duplicate cultures from a representative of two separate experiments. The control cpm value for IPs was 298.
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Effects of Genistein, Staurosporine, and TPA on Pervanadate-Induced IP Generation
Table 1 shows that pervanadate-induced formation of IPs was inhibited by the tyrosine kinase inhibitor genistein in a dose-dependent manner. There was also some decrease in the response to histamine, although to a lesser degree. The protein kinase inhibitor staurosporine inhibited the response to pervanadate at doses that did not inhibit the histamine response and greatly enhanced the thrombin response.
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Short-term incubation with the phorbol ester TPA (100 ng/mL) has been shown to inhibit thrombin-mediated generation of IPs in endothelial cells.1 As shown in Table 2, pretreatment with TPA reduced the IP response to thrombin by half but did not affect the IP response induced by pervanadate.
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Effects of Pervanadate on the Production of IPs and PGI2 Induced by Thrombin or Histamine
To investigate the possibility that thrombin- or histamine-induced IP generation might be modulated by PTPase(s), we examined the effects of costimulation with pervanadate and thrombin or pervanadate and histamine on endothelial cells. Fig 4 depicts the different effects of various doses of pervanadate on the generation of IPs induced by the agonists. A response ratio was calculated as follows:
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).
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, thrombin and pervanadate; 
, histamine and pervanadate) was measured by radioimmunoassay. Data are expressed as response ratio calculated as described in the "Results" section and represent mean±SEM from four experiments, each done in duplicate. Values (cpm, IPs) for control, histamine, and thrombin in a representative experiment were 110, 1851, and 979, respectively.
Tyrosine Phosphorylation
Fig 5a illustrates the effects of pervanadate on tyrosine phosphorylation in endothelial cells. There was some tyrosine phosphorylation detected in untreated cells, but it was gradually enhanced by doses up to 100 µmol/L pervanadate. Fig 5b shows the time response and the effects of the inhibitors genistein and staurosporine on tyrosine phosphorylation. An increase was detected after exposure to pervanadate for 4 minutes, and the phosphorylation continued to increase up to at least 20 minutes' exposure. Both genistein and staurosporine inhibited the pervanadate-induced tyrosine phosphorylation in a dose-dependent manner.
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As shown in Fig 6 thrombin alone caused only minimal tyrosine phosphorylation, which was greatly enhanced by cotreatment with pervanadate. Cotreatment with histamine and pervanadate resulted in a similar pattern of tyrosine phosphorylation to that obtained with pervanadate alone.
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Effects of Stimulation on Tyrosine Phosphorylation of Specific Proteins
Fig 7a shows the effect on tyrosine phosphorylation after 5 minutes' stimulation with pervanadate (20 µmol/L), histamine (33 µmol/L), or pervanadate and histamine together. Fig 7b shows the same blot after stripping and reprobing with antibodies against MAP kinase (p44) and PLC1. Mobility shift involving most of the MAP kinase band occurred after pervanadate stimulation, whereas only a small portion of the band is shifted after histamine stimulation. After cotreatment with pervanadate and histamine, all the MAP kinase band shifted mobility. In the blot probed with anti-phosphotyrosine (Fig 7a) there is a distinct new band present with the molecular weight of MAP kinase after stimulation with pervanadate. A faint band of the same molecular weight also appears after histamine stimulation. Pervanadate caused tyrosine phosphorylation of a protein with similar molecular weight to the protein detected by the PLC1 antibody (Fig 7a and 7b). Fig 7c shows the effect of stimulation with thrombin (3 U/mL) for 10 minutes on tyrosine phosphorylation. The results are similar to those seen with histamine.
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Pervanadate-Induced Tyrosine Phosphorylation of PLC1
Pervanadate-induced tyrosine phosphorylation of PLC1 and how it is affected by genistein were demonstrated after immunoprecipitation with antibodies against PLC1. As shown in Fig 8a, tyrosine phosphorylation of PLC1 increased with time up to at least 20 minutes. This response was similar to the time course of IP generation in pervanadate-treated cells (Fig 3
). As shown in Fig 8b, genistein inhibited pervanadate-induced tyrosine phosphorylation of PLC1 at the higher doses (50 and 100 µmol/L) but not at the low dose (20 µmol/L). This dose dependency for genistein is also similar to that for pervanadate-induced IP formation (Table 1).
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Tyrosine Phosphorylation of PLC1 After Cotreatment With Thrombin and Pervanadate Versus Histamine and Pervanadate
After stimulation with thrombin or histamine alone, no tyrosine phosphorylation of PLC1 was detected in the anti-PLC1 immunoprecipitate (Fig 9). The responses to cotreatment with thrombin and pervanadate versus histamine and pervanadate were different. While the tyrosine phosphorylation of PLC1 was enhanced after cotreatment with thrombin and pervanadate compared with that after pervanadate alone, costimulation with histamine and pervanadate caused no more tyrosine phosphorylation of PLC1 than did stimulation with pervanadate alone.
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Discussion |
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In this article we demonstrate that tyrosine phosphorylation of PLC
1 represents an alternative pathway for activation of human umbilical vein endothelial cells in a short-term reaction such as thrombin-induced production of PGI2. When the cells were exposed to the PTPase inhibitor pervanadate, they responded by increasing protein tyrosine phosphorylation of multiple cellular proteins, including PLC1, by increasing levels of IPs, releasing arachidonic acid, and producing PGI2. We also report on differences in the response of endothelial cells to combined treatment with thrombin and pervanadate in comparison to that of histamine and pervanadate. Although tyrosine phosphorylation of PLC1 could not be detected after stimulation with thrombin alone, cotreatment with thrombin and pervanadate caused synergistic increase in both tyrosine phosphorylation of immunoprecipitated PLC1 and the production of IPs. No such synergism was found between pervanadate and histamine. Finally, we show that MAP kinase is tyrosine phosphorylated after stimulation of the cells with either pervanadate, thrombin, or histamine.
Pervanadate is known to induce tyrosine phosphorylation of several proteins, including PLC in platelets and myeloid cells.20 21 25 26 27 The proposed mechanism involves inhibition of tyrosine-specific phosphatases,20 21 28 resulting in an indirect stimulation of tyrosine kinases. Recently, Bowden and coworkers10 used pervanadate to demonstrate tyrosine phosphorylation in response to the P2Y- and P2U-purinoceptor agonists on the assumption that tyrosine phosphorylation could not be detected without inhibiting the PTPases.
In endothelial cells treated with pervanadate alone, we found a correlation between the formation of IPs and tyrosine phosphorylation, including that of PLC1.
The dose responses were similar (Figs 2 and 5a), as was the time course of activation (Figs 3, 5b, and 8a). Although the accumulation of 6-oxo-PGF1 leveled off while the accumulation of IPs continued, the IP production preceded the initiation of PGI2 production, suggesting a cause-and-effect relationship. The continued production of IPs after treatment with pervanadate is in contrast to the leveling off demonstrated after receptor agonist activation.29 Separately, H2O2 or vanadate had negligible effects, which is in accordance with several other studies,24 30 although others have reported activation of endothelial cells after oxidant treatment.31 32 33 34
To further evaluate the relationship between tyrosine phosphorylation and IP generation after pervanadate stimulation, tyrosine kinase inhibitors were used. We found that both responses could be inhibited by staurosporine at 0.5 µmol/L. In contrast, this dose of staurosporine potentiated the inositol production in response to thrombin. Although the mechanism for this enhanced response is not known, possible explanations include inhibitory effects of staurosporine on protein kinase C or activation of G-proteins as observed by Kanaho and coworkers.35 Genistein, a specific inhibitor of tyrosine-specific protein kinases,36 37 inhibited dose dependently both pervanadate-induced general tyrosine phosphorylation and phosphorylation of immunoprecipitated PLC1. As shown in Table 1, IP formation was also decreased after genistein treatment, although not to the same extent as the tyrosine phosphorylation.
We have investigated the possible involvement of tyrosine phosphorylation in thrombin- or histamine-induced IP formation. The receptors for thrombin and histamine both belong to the G-protein–linked receptor family.15 16 The finding that costimulation with histamine and pervanadate resulted in an additive IP response is consistent with the generally accepted concept that G-proteins and tyrosine kinases activate distinct phosphatidylinositol-PLC isoenzymes. The synergistic effect of thrombin and pervanadate on IP formation indicates a more complex regulation of thrombin-induced endothelial signaling. Thrombin has been shown to stimulate tyrosine phosphorylation in endothelial cells.38 The thrombin receptor is not a tyrosine kinase and does not phosphorylate PLC directly. However, thrombin stimulation in endothelial cells could activate an intracellular PTK, which in turn might induce tyrosine phosphorylation and activation of PLC. Although we have not been able to detect PLC1 in the anti-phosphotyrosine immunoprecipitate obtained from cells stimulated with thrombin alone, costimulating the cells with thrombin and pervanadate resulted in an enhanced tyrosine phosphorylation of PLC1 (Fig 9). This indicates that activation of the thrombin receptor stimulates tyrosine phosphorylation of PLC, which can be detected only under conditions in which PTPases have been inhibited. The m5 muscarinic cholinergic receptor39 and the receptors for thrombin17 18 and angiotensin II,40 which all belong to the family of receptors containing seven-membrane–spanning domains, have been shown to stimulate tyrosine phosphorylation of PLC in other cell types, including platelets and vascular smooth muscle cells. Also, genistein has been shown to partially (40% to 50%) inhibit the IP response to bradykinin, whose receptor belongs to the G-protein receptor family, indicating that tyrosine phosphorylation may play a role in some phase of bradykinin-promoted IP formation.41 Paris and coworkers42 have shown that tyrosine kinase–activating growth factors, which alone are ineffective, potentiate thrombin or AlF4--induced phosphoinositide breakdown in hamster fibroblasts. They proposed that the growth factors might enhance the coupling between G-protein(s) and PLC, presumably through phosphorylation of one of these proteins. Recently, Daub and coworkers43 observed transactivation of the EGF receptor by activation of G-protein–coupled receptors, including the one for thrombin. Thus, there is rapidly evolving experimental support for a cross talk, detectable at the level of PLC, between the G-protein–dependent and the tyrosine phosphorylation–dependent pathways, at least in some cell types.
It has previously been shown that the PKC activator TPA inhibits IP formation after receptor activation with thrombin and also after stimulation with the G-protein activator AlF4- in human umbilical vein endothelial cells.9 Since AlF4- has its effects distal to the receptor, these results suggested that the inhibitory effects of TPA-activated PKC were at the level of a G-protein or PLC. Our present results (Table 2) show that the IP response induced by pervanadate is not affected by prior short-term treatment with TPA, further indicating that the TPA effects are mediated either through a G-protein or PLCß but not a tyrosine phosphorylation–dependent signal pathway. This finding is in agreement with previous results showing that phorbol esters distinguish between G-protein and tyrosine kinase pathways that are linked to phosphoinositide hydrolysis. While inhibiting IP generation induced by bombesin or vasopressin in Swiss 3T3 mouse cells, phorbol esters did not affect IP formation in response to PDGF.44
When endothelial cells were stimulated with either histamine or thrombin, an increase in tyrosine phosphorylation of MAP kinase was observed. MAP kinases (p42 and p44) are activated by phosphorylation on both tyrosine and threonine by the dual-recognition kinase MAP kinase kinase, which receives signals from both tyrosine kinase receptors and receptors coupled to G-proteins,45 although MAP kinase could be activated by Ca2+-independent mechanisms.46
Fleming and coworkers11 have recently shown Ca2+-dependent tyrosine phosphorylation of MAP kinase in human endothelial cells treated with bradykinin, histamine, or thaspigargin. In Chinese hamster ovary cells, activation of MAP kinase resulted in stimulation of cPLA2.47 An analogous pathway in endothelial cells could explain the synergistic effects on PGI2 production observed after costimulation with histamine and pervanadate, which did not act synergistically on IP production. The greater synergistic effects on PGI2 production observed when thrombin and pervanadate acted together most likely reflects the potentiating effects of pervanadate on IP generation induced by thrombin but not histamine (Fig 4).
In summary, stimulation of human umbilical vein endothelial cells with the PTPase inhibitor pervanadate leads to tyrosine phosphorylation of PLC1, generation of IPs, release of arachidonic acid, and PGI2 production. Cotreatment with thrombin and pervanadate but not histamine and pervanadate resulted in a synergistic increase in tyrosine phosphorylation of immunoprecipitated PLC1 and in a synergistic IP production. The results indicate that tyrosine phosphorylation of PLC1 represents an alternative pathway for activation of human umbilical vein endothelial cells in a short-term reaction such as thrombin-induced production of PGI2. The cascade of events from receptor activation to activation of PLC1 is not yet resolved, but the differential responses to thrombin compared with histamine suggest a possible mechanism by which the endothelium can respond to a large variety of signals in a specific way.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by the Research Fund of the University of Iceland, the Research Fund of the National University Hospital, and the Icelandic Science Council. We thank Arndis Theodors for skilled technical assistance.
Received August 24, 1995;
revision received June 7, 1996;
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