Surface-retained tPA is essential for effective fibrinolysis on vascular endothelial cells.

In a previous study, we demonstrated unique secretory dynamics of tissue plasminogen activator (tPA) in which tPA was retained on the cell surface in a heavy chain-dependent manner after exocytosis from secretory granules in vascular endothelial cells. Here, we examined how retained tPA expresses its enzymatic activity. Retained tPA effectively increased the lysine binding site-dependent binding of plasminogen on the cell surface and pericellular area; this was abolished by inhibition of enzymatic activity of either tPA or plasmin, which suggests that de novo generation of carboxyl-terminal lysine as a consequence of degradation of surface/pericellular proteins by plasmin is essential. Retained tPA initiated zonal clot lysis of a fibrin network that had been formed on vascular endothelial cells, which was preceded by the binding of plasminogen to the lysis front. Our results provide evidence that secreted and retained tPA is essential for maintaining both high fibrinolytic activity and effective clot lysis on the vascular endothelial cell surface.

Multimodal function of the sweet taste receptor expressed in pancreatic ß-cells: generation of diverse patterns of intracellular signals by sweet agonists.

The sweet taste receptor is expressed in the taste bud and is activated by numerous sweet molecules with diverse chemical structures. It is, however, not known whether these sweet agonists induce a similar cellular response in target cells. Using MIN6 cells, a pancreatic ß-cell line expressing endogenous sweet taste receptor, we addressed this question by monitoring changes in cytoplasmic Ca2+ ([Ca2+]i) and cAMP ([cAMP]i) induced by four sweet taste receptor agonists. Glycyrrhizin evoked sustained elevation of [Ca2+]i but [cAMP]i was not affected. Conversely, an artificial sweetener saccharin induced sustained elevation of [cAMP]i but did not increase [Ca2+]i. In contrast, sucralose and acesulfame K induced rapid and sustained increases in both [Ca2+]i and [cAMP]i. Although the latter two sweeteners increased [Ca2+]i and [cAMP]i, their actions were not identical: [Ca2+]i response to sucralose but not acesulfame K was inhibited by gurmarin, an antagonist of the sweet taste receptor which blocks the gustducin-dependent pathway. In addition, [Ca2+]i response to acesulfame K but not to sucralose was resistant to a Gq inhibitor. These results indicate that four types of sweeteners activate the sweet taste receptor differently and generate distinct patterns of intracellular signals. The sweet taste receptor has amazing multimodal functions producing multiple patterns of intracellular signals.

In vivo imaging analysis of the interaction between unusually large von Willebrand factor multimers and platelets on the surface of vascular wall.

To elucidate how unusually large von Willebrand factor (UL-VWF) multimers facilitate thrombus formation, their behavior was analyzed together with that of platelets in living mice deficient in the gene encoding the protease that cleaves UL-VWF, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13-/-). By crossing ADAMTS13-/- mice with green fluorescent protein-expressing transgenic mice (GFP mice), GFP-ADAMTS13-/- mice were obtained. The dynamics of GFP-expressing platelets were monitored employing intravital confocal fluorescent microscopy. Administration of a vasopressin derivative, DDAVP, a secretagogue of VWF increased the number of platelets adhered to vascular endothelial cells (VECs) on mesentery at sites recognized by an anti-VWF antibody. Some of these platelets were interconnected and aligned as beads on a string. They reached their maximum length at 5 min and were longer in GFP-ADAMTS13-/- mice than in GFP mice (5.3 ± 4.3, N = 6 vs 2.9 ± 2.1 µm, N = 4) (mean±SE). Focal injury of VECs by topical application of FeCl(3) developed longer (25, 3-50 vs 10, 2-25 µm, P < 0.01) (mean, 10th-90th percentile) and more stable (1.3, 0.3-6.3 vs 0.3, 0.2-1.3 s, P < 0.01) connected platelets in GFP-ADAMTS13-/- mice than in GFP mice. This study revealed that ADAMTS13 cleaves platelet-bound UL-VWF multimers, both during their secretion from VECs and after their adherence to injured vascular walls in veins. UL-VWF multimers either being secreted from VECs or circulating in plasma of ADAMTS13-/- mice appeared to facilitate the accumulation of longer and more stable VWF strings with more associated platelets on injured vascular walls.

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells.

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.

A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy.

Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

OBJECTIVE: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 µM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

Protein S attenuates the invasive potential of THP-1 cells by interfering with plasminogen binding on cell surface via a protein C-independent mechanism.

Protein S, a cofactor for activated protein C (aPC) to inactivate coagulation factors, also plays a pivotal role in inflammation. Based on our recent findings that aPC and protein S modifies tissue plasminogen activator (tPA)-catalyzed activation of Glu-plasminogen (Glu-plg), we analyzed possible role of protein S in cell-associated plasminogen activation and invasive potential of inflammatory cells. Monocyte-like THP-1 cells, to which both plasminogen and tPA bind, enhanced tPA-catalyzed plasminogen activation, which was partially abolished by protein S but not by aPC. Protein S attenuated both the plasminogen binding to THP-1 cells and associated their invasive potential through Matrigel.

Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)ß3 evokes phosphatidylserine exposure on their cell surface.

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)ß3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Preferential release of newly synthesized insulin assessed by a multi-label reporter system using pancreatic ß-cell line MIN6.

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic ß-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.

Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+) ([Ca(2+)](c)) and cAMP ([cAMP](c)) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+)](c). The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+)](c) response. The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor. Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.

Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo.

The exact mechanism of blood vessel thrombus formation remains to be defined. Here, we introduce a new approach to probe thrombus formation in blood vessels of living animals using intravital microscopy in green fluorescent protein (GFP)-transgenic mice to simultaneously monitor platelet aggregation and procoagulant activity. To this end, GFP-expressing platelets and annexin A5 labeled with a fluorescent dye were employed to visualize and analyze platelet aggregation and markers of procoagulant activity (platelet surface phosphatidylserine (PS)). Laser-induced thrombi increased and then decreased in size with time in vessels of living animals, whereas platelet surface PS initiated at the site of injury and then penetrated into the thrombus. PS-positive platelets were predominantly localized in the center of the thrombus, as was fibrin generation. The experimental system proposed here is a valuable tool not only for investigating mechanisms of thrombus formation but also to assess the efficacy of antithrombotic drugs within the vasculature.

Nafamostat attenuated the impairment of fibrinolysis in animal sepsis model by suppressing the increase of plasminogen activator inhibitor type 1.

BACKGROUND: In endotoxemia, plasminogen activator inhibitor-1 (PAI-1) increases and develops clinical symptoms by suppressing fibrinolysis. We analyzed therapeutic advantage of nafamostat, a broad-range protease inhibitor, on fibrinolysis in an animal sepsis model. METHODS: Male Wister rats infused with lipopolysaccharide (LPS) (50 mg/kg) alone or together with nafamostat (0.1 mg/kg/hr) for 4 hours were analyzed. RESULTS: Plasma PAI-1 (4.2: 4.0-5.0 ng/mL, median and interquartile range) increased after LPS infusion (3700: 3400-4000), which was attenuated by nafamostat (2300: 2100-2600, p < 0.05). Fibrin(ogen) degradation products after LPS injection (173: 152-182 microg/mL) were further elevated by nafamostat (205: 205-228, p < 0.05), Nafamostat attenuated polymorphonuclear neutrophils infiltration in the liver, and tended to suppress plasma tumor necrosis factor-alpha levels. Nafamostat did not affect thrombin generation, platelet count, markers of liver and kidney function, and overall mortality. CONCLUSIONS: Nafamostat appeared to improve impaired fibrinolysis by suppressing the increase of PAI-1 in plasma, though it did not largely improve clinical parameters.

Glucagon-like peptide 1 activates protein kinase C through Ca2+-dependent activation of phospholipase C in insulin-secreting cells.

Although the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca(2+) signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca(2+) influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca(2+) signaling can activate PKCalpha and PKCepsilon at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCalpha and PKCepsilon from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca(2+)-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCepsilon inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCepsilon-(149-164) (antp-PKCepsilon) and a conventional PKC inhibitor, Gö-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCgamma and 2) translocation of PKCalpha-DsRed and PKCepsilon-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCalpha and PKCepsilon, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.

Plasminogen activator inhibitor type 1 promotes fibrosarcoma cell migration by modifying cellular attachment to vitronectin via alpha(v)beta(5) integrin.

Both urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) are associated with a poor prognosis in cancer patients. We demonstrate that PAI-1 inhibits human fibrosarcoma cell (HT-1080) adhesion to vitronectin (Vn) via alpha (v)beta (5) integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cells attached more strongly to Vn and Col than to fibronectin (Fn), whereas PAI-1 interfered with cell attachment to Vn only. An integrin antagonist, RGD peptide, and anti-alpha (v)beta (5) integrin antibodies, which similarly inhibited cell attachment to Vn, also stimulated cell migration from Vn toward Col. u-PA did not modify cell attachment directly, but reversed the PAI-1-mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus HT-1080 cell migration appears to be modified by u-PA and PAI-1, altering cell adhesion to Vn via alpha (v)beta (5) integrin. This may be related to their tumor-promoting effect.

Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic beta cells.

The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium-evoked insulin secretion. In rat beta cells transfected with green fluorescent protein-tagged PKC-alpha (PKC-alpha-EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca(2)(+)](c)), which was accompanied by transient translocation of PKC-alpha-EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC-theta-EGFP, the C1 domain of PKC-gamma and PKC-epsilon-GFP. A high concentration of glucose induced repetitive elevation of [Ca(2)(+)](c) and repetitive translocation of PKC-alpha-EGFP. Diazoxide completely blocked both elevation of [Ca(2)(+)](c) and translocation of PKC-alpha-EGFP. We then studied the role of conventional PKC in calcium-evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Go-6976, an inhibitor of conventional PKC, and PKC-alpha pseudosubstrate fused to antennapedia peptide (Antp-PKC(19-31)) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö-6976 and Antp-PKC(19-31). However, when islets were stimulated for 60 min with high glucose, both Gö-6976 and Antp-PKC(19-31) reduced glucose-induced insulin secretion. Similar results were obtained by transfection of dominant-negative PKC-alpha using adenovirus vector. Taken together, PKC-alpha is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization-induced insulin secretion per se, but it also augments glucose-induced secretion.

Decoding of short-lived Ca2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C.

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca(2+) channels eliciting Ca(2+) influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca(2+) influx alone can activate PKC. The present study was conducted to investigate the Ca(2+) influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCalpha) and novel PKC (nPKC; PKCtheta), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCalpha to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca(2+) influx in the DsRed-tagged PKCalpha and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca(2+) influx can generate diacylglycerol (DAG), because cPKC is activated by Ca(2+) and DAG. We showed this in three different ways by demonstrating: 1) Ca(2+) influx-induced translocation of GFP-tagged C1 domain of PKCgamma, 2) Ca(2+) influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca(2+) influx-induced translocation of GFP-tagged PKCtheta, as a marker of DAG production and/or nPKC activity. Thus, Ca(2+) influx alone via voltage-dependent Ca(2+) channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca(2+).