Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Tactile allodynia initiated by local subcutaneous endothelin-1 is prolonged by activation of TRPV-1 receptors

Subcutaneous endothelin-1 (ET-1; 200 microM, 2 nmoles/paw) injected into the rat hind paw, has been shown to cause robust hind paw flinching (HPF) and paw licking, and to induce impulses selectively in primary nociceptors. Here we report that a much lower [ET-1] sensitizes the paw to a nocifensive withdrawal response to tactile stimulation (by von Frey hairs, VFH), a sensitization that involves local TRPV1 receptors. Injection of 10 microM ET-1 (0.1 nmole/paw) causes only marginal HPF but rapidly (20 mins after injection) lowers the force threshold for paw withdrawal (PWT) to VFH, to approximately 30% of pre-injection baseline. Such tactile allodynia persists for 3 hrs. In rats pre-injected with the TRPV1-antagonists capsazepine (CPZ; 1.33 mM) or 5'-iodoresiniferatoxin (I-RTX; 0.13 microM), 15 min before ET-1, a fast initial drop in PWT, as with ET-1 alone, occurs (to 40% or to 19% of baseline, respectively), but this earliest reduction then regresses back to the pre-injection PWT value more rapidly than with ET-1 alone. The recovery of allodynia from the maximum value is about two times faster for ET-1+CPZ and about 4 times faster for ET-1+ I-RTX, compared with that from ET-1 +vehicle (t(1/2) = 130, 60, and 250 mins, respectively). In contrast, spontaneous pain indicated by overt HPF from ET-1 is not attenuated by TRPV1 antagonists. Tactile allodynia is similarly abbreviated by antagonists of both ET(A) (BQ-123, 32 nmoles/paw) and ET(B) (BQ-788, 30 nmoles/paw) receptors, whereas HPF is abolished by this ET(A) antagonist but enhanced by the ET(B) antagonist. We conclude that low ET-1 causes tactile allodynia, which is characterized by a different time-course and pharmacology than ET-1-induced nociception, and that local TRPV1 receptors are involved in the maintenance of this ET-1-induced allodynia but not in the overt algesic action of ET-1.     

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.     

Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK

The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.     

Endothelin-1 inhibits adiponectin secretion through a phosphatidylinositol 4,5-bisphosphate/actin-dependent mechanism

Adiponectin is an adipokine with profound insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties. Plasma levels of adiponectin are reduced in insulin resistant states such as obesity, type 2 diabetes and cardiovascular disease. However, the mechanism(s) by which adiponectin concentrations are decreased during disease development is unclear. Studies have shown that endothelin-1 (ET-1), a vasoconstrictor peptide, affects adipocyte glucose metabolism and secretion of adipokines such as leptin, resistin, and adiponectin. The goal of our study was to determine the mechanism by which ET-1 decreases adiponectin secretion. 3T3-L1 adipocytes were treated for 24h with ET-1 (10nM) and then stimulated with vehicle or insulin (100 nM) for a period of 1-2h. Chronic ET-1 (24h) treatment significantly decreased basal and insulin-stimulated adiponectin secretion by 66% and 47%, respectively. Inhibition of phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis by the PLCbeta inhibitor, U73122, or exogenous addition of PIP(2):histone carrier complex (1.25:0.625 microM) ameliorated the decrease in basal and insulin-stimulated adiponectin secretion observed with ET-1. However, treatment with exogenous PIP(2):histone carrier complex and the actin depolymerizing agent latrunculin B (20 microM) did not reverse the ET-1-mediated decrease in adiponectin secretion. In conclusion, we demonstrate that ET-1 inhibits basal and insulin-stimulated adiponectin secretion through PIP(2) modulation of the actin cytoskeleton.     

Interaction of hCLIM1, an enigma family protein, with alpha-actinin 2

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein-protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36 kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with alpha-actinin 2, the skeletal muscle isoform of alpha-actinin. hCLIM1 protein was shown to interact with alpha-actinin 2 by yeast two-hybrid screening and immunochemical analyses. Yeast two-hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF-hand region of alpha-actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with alpha-actinin at the Z-disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton.     

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.     

Intrapericardial angiotensin II stimulates endothelin-1 and atrial natriuretic peptide formation of the in situ dog heart

Angiotensin (AT) II, endothelin (ET)-1, and atrial natriuretic peptide (ANP) play an important role in cardiovascular regulatory processes under physiologic and pathophysiologic conditions. All of these agents are present in the pericardial fluid, and alteration of their pericardial concentrations mirror changes in the myocardial interstitium. Moreover, the composition the pericardial fluid may also reflect the myocardial interaction of these agents. The local myocardial effects of AT II on cardiac ET-1 and ANP production, as well as on cardiovascular function, was studied by intrapericardial (ip) administration of AT II (0.125-1.0 microg/kg) to the in situ dog heart (n = 8). Big ET, ET-1, and ANP [1-28] fragment concentrations were measured by enzyme-linked immunosorbent assay in pericardial infusate samples and in peripheral blood before and after an AT II treatment of 15 mins. Systemic blood pressure (BP), heart rate (HR), and left ventricular contractility (dP/dt) were also recorded. In our studies, the pericardial big ET (but not ET-1) concentration was increased to a maximum value of 139 +/- 28 versus 74 +/- 12 pg/ml (control; P < 0.02) with ip AT II administration, with parallel elevations of the pericardial ANP levels (36.8 +/- 7.2 vs. 24.4 +/- 3.6 ng/ml; P < 0.05). The ip administration of AT II did not influence HR, and it elicited moderate changes in BP (BP(max), +14 +/- 2 mm Hg, P < 0.001; dP/dt(max), +10 +/- 3%, P < 0.02). The plasma levels of big ET, ET-1, and ANP did not change significantly. The results suggest that AT II promotes production of big ET and ANP in the heart. However, no detectable conversion of big ET-1 to ET-1 was observed within 15 mins. The myocardial formation of big ET-1 and ANP occurred, at least in part, independently of the changes in cardiovascular function.     

Phospholipase C-delta1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1

Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.     

The calpain 1-alpha-actinin interaction. Resting complex between the calcium-dependent protease and its target in cytoskeleton.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.     

The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.     

Interaction of fibronectin-coated beads with attached and spread fibroblasts : Binding, phagocytosis, and cytoskeletal reorganization

After 15 min incubations, binding of 0.8-, 6-, and 16-microns fibronectin-coated latex beads occurred primarily at the margins of chick embryo fibroblasts that previously were attached and spread on fibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-micron beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and alpha-actinin patches adjacent to the sites where the beads were bound. The formation of alpha-actinin patches could be detected with 6- or 16-microns beads shortly after initial bead binding to the cells, but a similar reorganization of alpha-actinin in response to the binding of 0.8-micron beads was not detected. The patches of alpha-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy (SEM) to be sites of cell interaction with 6- but not 0.8-micron beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-microns beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interaction.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

Phosphoinositide binding to the substrate regulates susceptibility to proteolysis by calpain

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein alpha-actinin to proteolysis by calpains 1 and 2. At first, alpha-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) binding to alpha-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave alpha-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P(3) binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of alpha-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P(3), alpha-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of alpha-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P(3)-mediated calpain proteolysis of alpha-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P(3) binding is a critical determinant for alpha-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.     

Effects of endothelin-1 and flunarizine on human trabecular meshwork cell contraction

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism because they exhibit smooth muscle-like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intraocular pressure (IOP) control. We propose an in vitro culture model as a method for the study of ET-1-induced human TM (HTM) cell contractility and for the study of whether pre-incubation with flunarizine, a calcium-channel blocker, can inhibit the action of ET-1. Experiments were performed on semiconfluent HTM cells (primary cultures established from normotensive human donor eyes) at the second passage, with phosphate-buffered saline (PBS) as a control. The contractile status of the cells was evaluated by a morphometric analysis of cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction, rather than regulatory volume decrease. After incubation with 10 microM ET-1 for 5 mins, we observed a reduction of HTM cell area with respect to PBS-treated cells: 2425 +/- 876 microm2 versus 3125 +/- 987 microm2 (P < 0.001); and cells exhibited a retraction in shape and a reduction in number of indented profiles. Administration of ET-1 at progressively lower doses produced a corresponding lower reduction of HTM cell area, suggesting a dose-response effect of ET-1. Pre-incubation with 10 microM flunarizine strongly inhibited the ET-1 effect on HTM cell contraction: 2806 +/- 865 microm2 versus 2910 +/- 846 microm2 (P = not significant). Our data indicate that ET-1 induced a statistically significant reduction in the area of HTM cells versus controls, and that ET-1 can directly influence the aqueous outflow. Moreover, we observed that flunarizine inhibited the effect of ET-1 on the HTM cells.     

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.     

alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Herein we report that alpha-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with alpha-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and alpha-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and alpha-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of alpha-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity.     

Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown

The aim of these experiments was to investigate whether inositol lipids might mediate some of the effects of extracellular matrix (ECM) on cellular form and functions. The lipid phosphatidylinositol bisphosphate (PIP2) plays a role in cytoskeletal regulation while its hydrolysis products, diacylglycerol and inositol triphosphate, serve as second messengers. We therefore measured the effect of adhesion to fibronectin (FN) on PIP2 and its hydrolysis products, in the presence and absence of the soluble mitogen PDGF. PDGF induced a threefold increase in release of water-soluble inositol phosphates in C3H 10T1/2 fibroblasts when cells were attached to FN, but had little effect in suspended cells. Suppression of inositol phosphate release in unattached cells was not due to dysfunction of the PDGF receptor or failure to activate phospholipase C-gamma; PDGF induced similar tyrosine phosphorylation of PLC-gamma under both conditions. By contrast, the total mass of phosphatidylinositol bisphosphate (PIP2), the substrate for PLC-gamma, was found to decrease by approximately 80% when cells were detached from their ECM attachments and placed in suspension in the absence of PDGF. PIP2 levels were restored when suspended cells were replated on FN, demonstrating that the effect was reversible. Furthermore, a dramatic increase in synthesis of PIP2 could be measured in cells within 2 min after reattachment to FN in the absence of PDGF. These results show that FN acts directly to stimulate PIP2 synthesis, and that it also enhances PIP2 hydrolysis in response to PDGF. The increase in PIP2 induced by adhesion may mediate some of the known effects of FN on cell shape and cytoskeletal organization, while regulation of inositol lipid hydrolysis may provide a means for integrating hormone- and ECM-dependent signaling pathways.