Lactosylceramide stimulates aortic smooth muscle cell proliferation.

We have investigated the effects of various sphingolipids on aortic smooth muscle cell proliferation employing viable cell counting, [3H] thymidine incorporation into DNA and the release of lactate dehydrogenase. Assays for UDP Gal: GlcCer Bl-4 galactosyltransferase (GalT-2) in control and treated cells were pursued simultaneously. Lactosylceramide stimulated cell proliferation in the order of 5 fold. Antibody against LacCer but not GbOse3Cer blocked the proliferative effects of LacCer in these cells. This phenomena was specific for aortic smooth muscle cells as LacCer decreased cell viability of aortic endothelial cells and had no effect on pulmonary endothelial cells. D-PDMP inhibited the activity of GalT-2 in smooth muscle cells and markedly prevented cell proliferation. In contrast, L-PDMP stimulated the activity of GalT-2 in smooth muscle cells and stimulated cell proliferation. Antibody against GalT-2 inhibited cell proliferation. Our findings suggest that the activation of GalT-2 leads to increased LacCer levels, which in turn, may be involved in aortic smooth muscle cell proliferation.     

Oxidized phospholipids in minimally modified low density lipoprotein induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells

Oxidized phospholipids, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), typically present in minimally modified low density lipoprotein, have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses (inflammation, proliferation, or cell death). It was the aim of this study to understand their impact on intracellular signal transduction pathways that are responsible for these biological effects. We found that in arterial smooth muscle cells, PGPC and POVPC activated sphingomyelinases, in particular the acid isoform, which is known to participate in the very early phase of apoptotic stress responses. In addition, mitogen-activated protein kinases, which are involved in induction of stress response and apoptosis were phosphorylated (activated). Finally, activation of caspase 3 was observed, showing that stimulation of smooth muscle cells with POVPC and PGPC is associated with apoptosis. Stimulation of all these enzymes by the oxidized phospholipids almost perfectly matched their activation by minimally modified LDL. Consequently, these phospholipids seem to be responsible for the effect of this particle on cell signaling. Survival and proliferation pathways including NF-kappa B or AKT kinase were not induced by POVPC and PGPC. Experiments with a specific inhibitor of acid sphingomyelinase named NB6 showed that this enzyme plays a central role in mediating the apoptotic effects of the oxidized lipids. Thus, we conclude that modified phospholipids induce signaling cascades via activation of acid sphingomyelinase finally leading to apoptosis of smooth muscle cells, which is a detrimental process in the development of atherosclerosis.     

A novel role of lactosylceramide in the regulation of tumor necrosis factor alpha-mediated proliferation of rat primary astrocytes. Implications for astrogliosis following neurotrauma

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.     

Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDP Gal:GlcCer: β1 → 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells

Recent molecular cloning studies have suggested the presence of at least two β4Gal transferase genes (β4GalT-V and β4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-α converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion [1]. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro⁻5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, β1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5–5 min of incubation with PDGF in both wild type and Pro⁻5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p⁴⁴MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p⁴⁴ MAPK activation and cell proliferation in Pro⁻5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, β4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p⁴⁴MAPK activation, phosphorylation of Akt and cell proliferation.     

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.     

Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase

We have investigated the effects of modifying LDL by Cu++ and various hemoglobin preparations on aortic smooth muscle cell proliferation and on the activation of mitogen activated protein kinase. We found that at very low concentrations (10 g/ml), LDL modified by all of the above agonists markedly stimulated cell proliferation (5–10 fold). This was accompanied by a 2–3 fold stimulation in mitogen activated protein kinase (MAPK) activity. We conclude that modification of LDL under situations that are closer to those found in vivo (i.e. hypoxic conditions), may involve the activation of MAPK as a common biochemical mechanism of action. This in turn, contributes to aortic smooth muscle cell proliferation.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages

The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.     

Oxidized Phospholipids Induce Ceramide Accumulation in RAW 264.7 Macrophages: Role of Ceramide Synthases

Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC) are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs). These OxPLs are factors contributing to pro-atherogenic effects of oxidized LDLs (OxLDLs), including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer) which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C₁₆-, C₂₂-, and C_24:0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.     

The oxidized phospholipids POVPC and PGPC inhibit growth and induce apoptosis in vascular smooth muscle cells

Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are typically present in oxidatively modified low density lipoprotein (oxLDL) and have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses like inflammation, proliferation, or cell death. The aim of this study was to elicit the type and outcome of the cellular response of vascular smooth muscle cells (VSMC) upon treatment with POVPC and PGPC. Both oxidized phospholipids led to inhibition of cell proliferation and showed cytotoxic effects in VSMC. Several morphological criteria, the presence of typical DNA fragments, and a phosphatidylserine shift towards the outer leaflet of the cell membrane revealed that apoptosis was the predominant mode of cell death. In all experiments, POVPC was found to be a more potent inducer of apoptosis than PGPC. Interestingly, in the presence of high levels of serum in the growth media the proapoptotic but not the antiproliferative effects of both oxidized phospholipids were abolished. Thus, we conclude that under low serum conditions both intact POVPC and PGPC are proapoptotic mediators. Under high serum conditions, these lipids are hydrolyzed and the resultant lipid mixture containing the degradation products is no longer apoptotic but antiproliferative. Thus, the direct and indirect effects of POVPC and PGPC on cell viability may account for the detrimental actions of oxLDL on VSMC.     

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids. LDL internalization is not required for cellular lipid uptake. Collectively, our data provide evidence that oxidized phospholipids, owing to their high exchangeability between lipoproteins and cell membranes, may act within a short time on different cellular sites in VSMCs and affect various lipid and protein components through physical or chemical interactions, which might then serve as starting points for intracellular signaling.     

Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation

We have investigated the effects of oxidized low density lipoproteins(Ox-LDL) on aortic smooth muscle cell (SMC) proliferation andthe biosynthesis of glycosphingo-lipids. We found that Ox-LDL exerted a concentration, time, and temperaturedependent alteration of cell proliferation and the biosynthesisof lactosylceramide. At low concentrations (5–10 µg/mlmedium) Ox-LDL stimulated cell proliferation measured by anincrease in the incorporation of ³H-thymidine in cells and thesynthesis of lactosylceramide, but not glucosylceramide synthesis.Oxidized LDL exerted a threefold increase in the incorporationof [³H]-galactose and [³H]-serine in lactosylceramide. The activityof lactosylceramide synthetase; UDP-galactose glucosylceramideß1     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues

We have measured the levels of glycosphingolipids and the activityof glycosphingolipid glycosyltransferases in human aortic intimaand media from patients who died of atherosclerosis. The effectsof lactosylceramide (LacCer) and glucosylceramide (GlcCer) fromplaque intima on smooth muscle cell proliferation were assessed.When the GIcCer data was expressed as (pg GlcCer/mg cholesteroland/mg total phospholipid, a 28-fold and 7-told increase inplaque intima compared to normal intima was observed. Similarly,the level of LacCer was elevated 5-fold and 4-fold, respectively,compared to unaffected intima. The activity of UDPGicCer: ceramideß1     

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl⁻ channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl⁻ channel in H₂O₂-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H₂O₂-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl⁻ channel can modulate H₂O₂-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.     

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT₁) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [³H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [¹²⁵I]AngII to AT₁ receptors was blocked by Ib in a concentration-dependent manner with IC₅₀ value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.