CCK-stimulated changes in pancreatic acinar morphology are mediated by rho

BACKGROUND/AIMS: Treatment of isolated pancreatic acini with high concentrations of cholecystokinin (CCK) is known to induce rapid changes in the cellular morphology. The signalling pathways remain to be characterized. METHODS: Pancreatic acini were permeabilized by digitonin and incubated with various agents. The acinar morphology was investigated by microscopy. The activation of p125 focal adhesion kinase was determined by Western blot analysis. Amylase was measured photometrically. RESULTS: The functionality of the permeabilized acini was tested by measuring stimulated amylase release. 300 microM GTP gamma S was almost as efficient as CCK to stimulate amylase release, while 300 microM GDP beta S inhibited the CCK-stimulated amylase release. Stimulation of permeabilized acini with 0.1 microM CCK induced similar morphological changes as in unpermeabilized acini. Incubation of permeabilized acini with GTP gamma S mimicked the CCK-induced changes, whereas a preincubation with GDP beta S prevented the CCK effects on the acinar morphology. Inhibition of the small G protein rho, which activates p125 focal adhesion kinase, by Clostridium botulinum C3 transferase also prevented the CCK-stimulated morphological changes. Preincubation of intact acini with cell-permeable inhibitors of protein kinase C, MEK or p38MAPK, or with the intracellular calcium chelator BAPTA/AM was without significant effect on the CCK-stimulated changes. CONCLUSION: The CCK-induced morphological changes seem to be mediated by G protein signalling via the small G protein rho and the associated activation of p125 focal adhesion kinase.     

External calcium facilitates signalling, contractile and secretory mechanisms induced after activation of platelets by collagen

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca2+. We have studied the involvement of exogenous Ca2+ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca2+ levels ([Ca2+]i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca2+ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca2+, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca2+]i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca2+. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca2+, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca2+ depends on a functional cytoskeleton.     

Tumor necrosis factor-alpha mediates pancreatitis responses in acinar cells via protein kinase C and proline-rich tyrosine kinase 2

BACKGROUND & AIMS: Although tumor necrosis factor alpha is implicated as an important mediator of the inflammatory response in acute pancreatitis, its role in other pathologic features of the disease remains unknown. We investigated the role for tumor necrosis factor alpha in cytoskeletal responses and the underlying signaling mechanisms in pancreatic acinar cells. METHODS: In isolated rat pancreatic acini and AR42J cells, we determined the effect of tumor necrosis factor alpha on the actin cytoskeleton by rhodamine-phalloidin. Using pharmacological and molecular approaches, we assessed the involvement of protein kinase C, Src kinases, and proline-rich tyrosine kinase 2 in the process. We also studied the involvement of these signaling pathways in tumor necrosis factor-alpha-induced nuclear factor-kappaB activation and apoptosis. RESULTS: Tumor necrosis factor-alpha increased the tyrosine phosphorylation of proline-rich tyrosine kinase 2 in acinar cells. The broad-spectrum protein kinase C inhibitor and the Src kinase inhibitor both inhibited tumor necrosis factor-alpha-induced proline-rich tyrosine kinase 2 phosphorylation, but at different tyrosine residues. Using protein kinase C isoform-specific inhibitors and the antisense approach, we showed that protein kinase C delta and mediate proline-rich tyrosine kinase 2 tyrosine phosphorylation. Tumor necrosis factor-alpha caused disorganization of the actin cytoskeleton by a mechanism dependent on protein kinase C, Src kinases, and proline-rich tyrosine kinase 2. Inhibition of protein kinase C, but not Src kinases, decreased tumor necrosis factor-alpha-induced apoptosis. Furthermore, with antisense transfections, we showed that protein kinase C delta and , but not proline-rich tyrosine kinase 2, mediate tumor necrosis factor alpha-induced nuclear factor-kappaB activation. CONCLUSIONS: Tumor necrosis factor-alpha activates proline-rich tyrosine kinase 2 to cause cytoskeletal disorganization and nuclear factor-kappaB to cause inflammatory response, and it triggers cell death signaling through divergent mechanisms mediated by protein kinase C. The results provide insights into the mechanisms in pancreatic acinar cells that link tumor necrosis factor alpha to critical processes in acute pancreatitis.     

Modification of actin,myosin and tubulin distribution during cytoplasmic granule movements associated with platelet adhesion

BACKGROUND AND OBJECTIVES: Cytoskeletal elements determine the changes in platelet cell shape which occur during adhesion, aggregation and release of granular contents as part of the activation process. The aim of this study was to characterize the changes in the distribution of actin filaments, myosin and tubulin molecules during several stages of platelet adhesion to glass and their association with granule displacement, as assessed by confocal microscopy. DESIGN AND METHODS: Platelets obtained from healthy donors were adhered to glass and cytoskeleton distribution was characterized and correlated to changes of cell shape and intracellular granule displacement by immunofluorescence assays and phase contrast microscopy. Treatment with specific cytoskeleton inhibitors such as cytochalasin D, butanedione monoxime and colchicine were used before and after the adhesion process. The spatial distribution of the cytoskeleton in association with cytoplasmic granules was analyzed in both confocal microscopy projections and three-dimensional images obtained by merging the respective projections. RESULTS: Our experiments revealed that as platelets contact the substrate, a sequential and simultaneous rearrangement of actin filaments, myosin and tubulin molecules occurred and this was related to cell shape, as well as to movements of cytoplasmic granules. Treatment of platelets with cytoskeleton inhibitors, modified not only the target molecule but also other cytoskeletal components with consequent alterations in the studied platelet functions. INTERPRETATION AND CONCLUSIONS: During platelet adhesion to glass and granule displacement, a close spatial and functional relation between actin filaments, myosin molecules and microtubules was observed suggesting that these different cytoskeleton components interact in supporting the platelet functions here studied.     

肥胖抑制素对大鼠胰腺腺泡和胰腺小叶淀粉酶分泌的影响

目的 研究不同剂量肥胖抑制素(Obestatin)对大鼠离体胰腺腺泡及胰腺小叶淀粉酶分泌的影响.方法 体外分离大鼠胰腺腺泡,与不同浓度Obestatin(0、0.1、1、10、30 nmol/L)共培养1 h;另一组加Obestatin 30 min后加入100 pmol/L的胆囊收缩素(CCK-8)继续培养30 min.分离含有胰腺内神经末端、胰岛细胞及外分泌细胞的胰腺小叶,与不同浓度Obestatin共培养30 min;另一组加Obestatin同时加75 mmol/L KC1共培养30 min.测定培养液及细胞或胰腺小叶组织内淀粉酶含量,计算淀粉酶分泌率.结果 0、0.1、1、10、30 nmol/L Obestatin不影响胰腺腺泡细胞淀粉酶分泌率[(3.48±1.44)％、(3.70±1.39)％、(3.36±1.24)％、(3.86±1.41)％、(4.54±2.01)％].CCK-8能显著刺激腺泡细胞的淀粉酶分泌率[(13.84±2.63)％比(3.48±1.44)％,P＜0.05],但0.1、1、10 nmol/L Obestatin 对CCK-8诱导的胰腺腺泡淀粉酶分泌无显著性影响[(14.55±1.70)％、(13.79±1.81)％、(14.39±1.12)％].Obestatin(0.1、1、10、30 nmol/L)不影响胰腺小叶淀粉酶分泌率.KCl可显著刺激胰腺小叶淀粉酶分泌率[为对照组的(1.84±0.29)倍,P＜0.05],但Obestatin对KC1诱导的胰腺小叶淀粉酶分泌无显著影响[为对照组的(2.01±0.30)、(1.89 ±0.41)、(1.74±0.14)、(1.88±0.33)倍].结论 Obestatin对体外培养的大鼠胰腺腺泡细胞及胰腺小叶淀粉酶分泌率无显著影响,也不能阻断或协同CCK-8和KCl对胰腺淀粉酶分泌的促进作用.     

Activation of cell adhesion kinase beta by mechanical stretch in vascular smooth muscle cells

We have studied whether activation of cell adhesion kinase beta (CAKbeta) is involved in stretch-induced signaling pathway in cultured rat vascular smooth muscle cells. Cyclic stretch (1 Hz) induced a rapid (within 1 min) phosphorylation of CAKbeta, whose effect was time and strength dependent. Both Ca(2+) and Na(+) ionophores (A23187 and monensin) stimulated phosphorylation of CAKbeta in a similar fashion to mechanical stretch. The stretch-induced phosphorylation of CAKbeta was inhibited completely by an intracellular Ca(2+) chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] and largely by gadolinium, but only partially by an extracellular Ca(2+) chelator (EGTA). An angiotensin type 1 receptor antagonist (CV11974) abolished the phosphorylation of CAKbeta stimulated by angiotensin II, but not by mechanical stretch. Mechanical stretch rapidly (within 1 min) increased the association of CAKbeta with c-Src, but not pp125(focal adhesion kinase). Stretch-induced phosphorylation of ERK1/2 was inhibited by EGTA and an inhibitor of the Src kinase family [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], but not by cytochalasin D, to disrupt actin polymerization. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or cytochalasin D did not affect stretch-induced phosphorylation of CAKbeta. These data suggest that mechanical stretch stimulates activation of CAKbeta, followed by its association with c-Src, which requires ion influx mainly via stretch-activated nonselective ion channels, thereby leading to activation of the p21(Ras)/ERK1/2 cascade in vascular smooth muscle cells.     

Inhibitory effect of somatostatin analog, SMS 201-995, on exocrine secretion from isolated rat pancreatic acini]

In vitro effect of somatostatin analog, SMS 201-995 (SMS), on pancreatic exocrine secretion was investigated using isolated rat pancreatic acini. SMS had no effect on basal, cholecystokinin octapeptide (CCK-8)- or secretin-stimulated amylase release. SMS inhibited pancreatic amylase release in response to simultaneous stimulation with secretin and CCK-8 in a dose-dependent manner. Significant inhibition was observed with 10 nM SMS and maximal inhibition with 0.1-1 microM SMS. Amylase release in response to the combination of 100 pM CCK-8, 1 nM secretin and 0.1-1 microM SMS was similar to that to 100 pM CCK-8 alone. Secretin significantly increased acinar cell cAMP content. SMS partially inhibited an increase in cAMP content induced by secretin. The present study has demonstrated, therefore, that SMS directly inhibits the potentiating effect of secretin on exocrine secretion in part by inhibiting an increase in secretin-induced cAMP accumulation in rat pancreatic acinar cells.     

Abstracts of papers presented at the Japanese–European Kobe Symposium on Thrombosis, 6–7 November 2000, Kobe Gakuin University,Kobe, Japan

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca²⁺. We have studied the involvement of exogenous Ca²⁺ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca²⁺ levels ([Ca²⁺]_i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca²⁺ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca²⁺, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca²⁺]_i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca²⁺. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca²⁺, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca²⁺ depends on a functional cytoskeleton.     

Expression and Cytoskeletal Association of Integrin Subunits Is Selectively Increased in Rat Perivenous Hepatocytes After Chronic Ethanol Administration

BACKGROUND: For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. METHODS: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. RESULTS: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. CONCLUSIONS: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.     

Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP- 280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb- IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb- IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb- IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.     

Selective involvement of p130Cas/Crk/Pyk2/c-Src in endothelin-1-induced JNK activation

Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds on which the G protein-coupled receptor (GPCR)-induced signaling complex might assemble. We have recently reported that Ca2+-sensitive tyrosine kinase, Pyk2, and epidermal growth factor receptor (EGFR) act as independently regulated scaffolds in cardiomyocytes. In this report, we investigated the activation and regulation of p130Cas, Crk, Pyk2, and c-Src by a well-known hypertrophic agonist, endothelin-1 (ET), and determined their contributions to the activation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in cardiomyocytes. Like Pyk2, ET-induced tyrosine phosphorylation of p130Cas was significantly inhibited by either chelating intracellular Ca2+ ([Ca2+]i) or a protein kinase C inhibitor, calphostin C. This activation of p130Cas was also abrogated by the tetrapeptide RGDS, which disrupts integrin heterodimerization; cytochalasin D, which depolymerizes the actin cytoskeleton; or a selective Src family kinase inhibitor, PP2, but not by an EGFR inhibitor, AG1478. We also observed ET-induced temporal associations of Pyk2 with active c-Src, followed by p130Cas with Pyk2, c-Src, and Crk. Overexpression of a dominant-negative mutant of p130Cas (CasDeltaSD), Crk (CrkSH2m), Pyk2 (PKM), or C-terminal Src kinase (Csk), but not of a deletion mutant of EGFR (533delEGFR), attenuated ET-induced JNK activation. Similarly, an ET-induced increase in c-jun promoter luciferase activity was inhibited by overexpression of CasDeltaSD, CrkSH2m, PKM, or Csk. In contrast, ET-induced ERK activation and c-fos gene expression were predominantly regulated by EGFR. Collectively, the focal adhesion-dependent p130Cas/Crk/Pyk2/c-Src-mediated pathway is selectively involved in ET-induced JNK activation in cardiomyocytes.     

Mechanical stretch has contrasting effects on mediator release from bronchial epithelial cells, with a rho-kinase-dependent component to the mechanotransduction pathway

INTRODUCTION: In vivo, the airway epithelium stretches and relaxes with each respiratory cycle, but little is known about the effect this pattern of elongation and relaxation has on bronchial epithelial cells. We have used a model of cell deformation to measure the effect of stretch on inflammatory cytokine release by the BEAS 2B cell line, and to examine the method of mechanotransduction in these cells. METHODS: BEAS 2B cells were cyclically stretched using the Flexercell system. IL-8 and RANTES protein and RNA levels were measured after different elongations, rates and duration of stretch. An inhibitor of Rho (Ras Homologous)-associated kinases was used, to assess the effect of blocking downstream of integrin signalling. Immunofluorescent staining of paxillin was used to study the effect of stretch on the distribution of focal contacts and the organisation of the actin cytoskeleton. RESULTS: IL-8 release by BEAS 2B cells was increased by cytokine stimulation and stretch, whereas RANTES levels in the cell supernatant decreased after stretch in a dose-, time- and rate-dependent manner. Thirty percent elongation at 20 cycles/min for 24h increased IL-8 levels by over 100% (P < 0.01). Blocking rho kinase using Y-27632 inhibited the effect of stretch on IL-8 release by the BEAS 2B cells. Immunofluorescent staining demonstrated that stretch caused dramatic disassembly of focal adhesions and resulted in the redistribution of paxillin to the peri-nuclear region. CONCLUSION: This study demonstrates a marked effect of stretch on bronchial epithelial cell function. We propose that stretch modulates epithelial cell function via the activation of rho kinases. The observation that stretch promotes focal adhesion disassembly suggests a mechanism whereby focal adhesion turnover (coordination of assembly and disassembly) is essential for mechanotransduction in bronchial epithelial cells.     

Cytochalasin D and E: effects on fibrinogen receptor movement and cytoskeletal reorganization in fully spread, surface-activated platelets: a correlative light and electron microscopic investigation.

This study investigates the involvement of actin microfilaments in fibrinogen receptor redistribution and cytoskeletal reorganization that takes place in fully spread, surface-activated platelets. Colloidal gold-labeled fibrinogen (Fgn-Au label) in conjunction with video-enhanced differential interference contrast light microscopy (VDIC) was used to identify fibrinogen binding sites, glycoprotein IIb/IIIb (GPIIb/IIIa), on fully spread platelets. Platelets were treated with cytochalasins D and E (5 x 10(-5) mol/L to 5 x 10(-8) mol/L) for 10 minutes, before or after incubation with Fgn-Au label. Results observed with VDIC were subsequently confirmed by high-voltage transmission and low voltage-high resolution scanning electron microscopic examination of the specimens. Preincubation of activated platelets with cytochalasin D or E (5 x 10(-5) and 5 x 10(-6) mol/L) inhibited fibrinogen receptor redistribution and abolished cytoskeletal reorganization in fully spread platelets. After surface-activated platelets were incubated with Fgn-Au label, treatment with the above concentrations of cytochalasin D or E disrupted cytoskeletal reorganization and caused random movement of previously redistributed receptor-ligand complexes. Incubation of platelets with cytochalasin E 5 x 10(-6) mol/L prevented platelet activation and spreading. Thus, actin filaments appear necessary for platelet spreading from the discoid to the fully spread stage. The ligand-triggered, cytoskeletally directed movement of fibrinogen receptors in fully spread platelets appears to be dependent on the presence of intact, polymerized actin. This movement is distinct from the cytochalasin-insensitive accumulation of GPIIb/IIIa-ligand in the channels of the open canalicular system.     

Stimulatory effect of cytochalasin D on antigen-induced phospholipase D activation in a murine mast cell model (RBL-2H3)

To investigate the possible involvement of cytoskeletal components in antigen (Ag)-mediated activation of phospholipase D (PLD) in rat basophilic leukemia (RBL-2H3) cells, the effects of cytochalasin D, which is known to interfere with actin organization in various cells, on Ag-induced PLD activity were examined. Cytochalasin D, at concentrations that induced distinct shape changes of RBL-2H3 cells, enhanced the Ag-induced 5-HT (serotonin) release and formation of phosphatidylbutanol (PBut), a specific and stable metabolite produced by PLD activity in a concentration-dependent manner. Concomitantly, Ag-induced 1,2-diacylglycerol (DG) accumulation as well as phosphatidic acid (PA) production were increased by the drug. In contrast, cytochalasin D had no effect on PLD activation in response to phorbolmyristate acetate, an activator of protein kinase C (PKC), and Ca²+ ionophore A23187. These results suggest that cytoskeletal components may modulate Ag-induced PLD activation upstream of PKC and Ca²+ in RBL-2H3 cells.     

Disruption of filamentous actin diminishes hormonally evoked Ca2+ responses in rat liver.

Previous studies have suggested a role for the actin cytoskeleton in hormonally evoked Ca2+ signaling in the liver. Here, we present evidence supporting a connection between filamentous actin (F-actin) organization and the ability of vasopressin and glucagon to increase cytosolic free-Ca2+ ([Ca2+]i) levels. F-actin was disrupted in hepatic cells by perfusion of rat liver with cytochalasin D. Epifluorescence microscopy of subsequently isolated cells showed reduced cortical fluorescent phalloidin staining in cytochalasin D-treated liver cells. Cytochalasin D pretreatment of liver cells reduced the vasopressin-stimulated elevation of [Ca2+]i by 60% and of glucagon by 50%. Experiments performed on cytochalasin D-treated cells using Mn2+ as an indicator of Ca2+ influx quenched fura-2 fluorescence signals following vasopressin administration. This indicates that a structurally intact cortical F-actin web is not a prerequisite for the influx of calcium. Therefore, the attenuation of the increase in cytosolic calcium observed in cytochalasin D-treated liver cells was likely caused either by the depletion of the calcium store by treatment with cytochalasin D or by the need for an intact cytoskeletal structure for its release. Because the resting level of calcium did not change in cells exposed to cytochalasin D, the latter is likely. The reduced [Ca2+]i response may be the mechanism by which cytochalasin D pretreatment inhibits vasopressin-induced metabolic effects. Cytochalasin D pretreatment also decreased the ability of glucagon to stimulate gluconeogenesis and reduced the stimulation of O2 uptake usually observed following glucagon administration. In conclusion, these results suggest that the hormonal elevation of [Ca2+]i and resultant activation of specific metabolic pathways require normal F-actin organization.     

Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal-regulated kinase (ERK) 1/2 and p70(S6K) pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70(S6K) pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II-dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II-induced ERK1/2 activation, but p70(S6K) activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II-induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II-induced FAK phosphorylation and ERK1/2 activation, but not p70(S6K) phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II-induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.     

Differential activation of p42ERK2 and p125FAK by cholecystokinin and bombesin in the secretion and proliferation of the pancreatic amphicrine cell line AR42J

Background: AR42J rat pancreatic acinar carcinoma cells have retained the potential to secrete digestive enzymes in addition to their ability to proliferate upon stimulation with regulatory peptides. We investigated the involvement of p42^ERK2 and p125^FAK (extracellular signal-regulated protein kinase and focal adhesion protein kinase, respectively) by cholecystokinin and bombesin stimulation with regard to secretion and mitogenesis. Methods: The p42^ERK2 activity was measured by kinase assay and the activation of p125^FAK by antiphosphotyrosine Western blot analysis of p125^FAK immunoprecipitates. The expression of both kinases was determined by Western blot analysis, the amylase secretion by colorimetry, and the DNA synthesis by [3H]thymidine incorporation. Results: p42^ERK2 and p125^FAK were activated by cholecystokinin and bombesin with maximum stimulation at concentrations above 10 nM. Bombesin was a weaker activator of p42^ERK2 and p125^FAK, causing only half of the kinase activity induced by stimulation with cholecystoki nin. PD98059 was shown to inhibit p42^ERK2, while tyrphostin 25 blocked p125^FAK tyrosine phosphorylation. Preincubation of AR42J cells with PD98059 or tyrphostin 25 was without influence on cholecystokinin- or bombe-sin-stimulated secretion in normal or 72-hour dexameth-asone-pretreated cells. In contrast, inhibition of both protein kinases leads to reduced cholecystokinin-stimulated [3H]thymidine incorporation rates. Conclusions: Cholecystokinin induced proliferation of AR42J cells by strong activation of p42^ERK2 and p125^FAK. Bombesin failed to stimulate DNA synthesis, probably due to its reduced potency to stimulate these kinases. Both protein kinases are not implicated in the process of enzyme secretion.     

A p160ROCK-specific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth

BACKGROUND/AIMS: p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Recently, Rho signaling pathways were reported to play an important role in the activation of rat hepatic stellate cells (HSC). The aim of this study was to investigate the mechanism of action of a p160ROCK-specific inhibitor, Y-27632, on cultured rat HSC. METHODS: HSC were isolated from normal rat livers and cultured on fibronectin-coated dishes. The cell morphology and actin cytoskeleton were studied with phase contrast and fluorescence microscopy, respectively. Immunoblot analysis was used to examine phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, and the expression of cell cycle-associated proteins. HSC proliferation was measured by quantitating the percentage of cells that exhibited nuclear incorporation of 5-bromodeoxyuridine. Type I collagen gene expression and accumulation in HSC culture media were evaluated by Northern blot and enzyme-linked immunosorbent assay, respectively. RESULTS: Y-27632 consistently blocked cell spreading and suppressed RhoA-induced formation of stress fibers in HSC. In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase. Cells treated with Y-27632 failed to proliferate, in contrast to untreated spread cells. This shape-dependent block in cell proliferation correlated with a failure to increase cyclin D1 protein level and to down-regulate the cell cycle inhibitor p27. Y-27632 decreased type I collagen gene expression and accumulation in HSC culture media. CONCLUSIONS: Our findings indicate that p160ROCK-mediated actin stress fiber assembly is involved in the pathophysiology of hepatic fibrogenesis and suggest that inhibitors of the RhoA-ROCK pathway might be useful therapeutically in liver fibrogenesis.     

beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.     

Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation

Abstract

Prostacyclin (PGI₂) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI₂. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI₂ and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI₂-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI₂ were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI₂. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI_2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.