Pregnancy-stimulated growth of vascular smooth muscle cells: Importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor

Dramatic smooth muscle cell (SMC) growth occurs in the uterine artery during pregnancy. The potential for pregnancy-associated growth may also exist at other vascular sites. We tested the hypothesis that increased growth of uterine artery SMC isolated from pregnant (vs. nonpregnant) guinea pigs would be detectable in culture, that pregnancy-associated phenotypic changes would also be found in nonuterine vascular cells (aortic SMC), and that the enhanced growth would be dependent on estrogen, peptide growth factors like platelet-derived growth factor (PDGF), and protein kinase C (PKC). Growth responses were measured by [³H]-thymidine incorporation and cell counts. Uterine artery SMC from pregnant guinea pigs grew to a higher plateau density with serum stimulation, had increased spontaneous DNA synthesis and persistent growth following serum withdrawal, and were more responsive to 3–30 ng/ml PDGF-BB than nonpregnant cells. Aortic SMC from pregnant animals also grew to a higher plateau density and had enhanced responsiveness to PDGF-BB. This increased response to PDGF-BB by pregnant uterine artery and aortic SMC (40–233% increase over nonpregnant PDGF result) was reproduced in nonpregnant cells by pretreatment for 1–24 h with 17-beta(β)-estradiol (30–100 nM). Neither the pregnancy-induced difference nor the estradiol pretreatment was associated with increased PDGF-BB binding activity. The synergistic effect of 17β-estradiol was partially (62%) reproduced with 17-alpha(α)-estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17β-estradiol modulates the PDGF-BB response by both estrogen-receptor- and nonreceptor-mediated mechanisms. To test if the estrogen effects were dependent on PKC, two different antagonist strategies (3 μM dihydrosphingosine and phorbol-ester-induced downregulation) were applied prior to 17α- or β-estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17β-estradiol on PDGF was then reproduced by 1 h pretreatment with the cell-permeable PKC activator, 10 nM PMA. We conclude that pregnancy stimulates increased growth of uterine and aortic SMC in vitro which is dependent on estrogen, PDGF, and PKC and may be important in vascular remodeling during pregnancy. © 1996 Wiley-Liss, Inc.     

Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o)

In this study, we examined theNa⁺-K⁺-Cl cotransporter activityand expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05).Expression of an ~161-kDa cotransporter protein was increased by 91%in the DBcAMP-treated astrocytes. Moreover, the specific[³H]bumetanide binding was increased by 67% in theDBcAMP-treated astrocytes. Inhibition of protein synthesis bycyclohexamide (2-3 µg/ml) significantly attenuated theDBcAMP-mediated upregulation of the cotransporter activity andexpression. The Na⁺-K⁺-Clcotransporter in astrocytes has been suggested to play a role inK⁺ uptake. In 75 mM extracellular K⁺concentration, the cotransporter-mediated K⁺ influx wasstimulated by 147% in nontreated cells and 79% in DBcAMP-treatedcells (P < 0.05). To study whether this highK⁺-induced stimulation of the cotransporter is attributedto membrane depolarization and Ca²⁺ influx, the role of theL-type voltage-dependent Ca²⁺ channel was investigated. Thehigh-K⁺-mediated stimulation of the cotransporter activitywas abolished in the presence of either 0.5 or 1.0 µM of the L-typechannel blocker nifedipine or Ca²⁺-free HEPES buffer. Arise in intracellular free Ca²⁺ in astrocytes was observedin high K⁺. These results provide the first evidence thatthe Na⁺-K⁺-Cl cotransporterprotein expression can be regulated selectively when intracellular cAMPis elevated. The study also demonstrates that the cotransporter inastrocytes is stimulated by high K⁺ in aCa²⁺-dependent manner.

     

CD40 signaling replaces CD4+ lymphocytes and its blocking prevents chronic rejection of heart transplants

Chronic rejection remains the major obstacle to long term survival in heart transplant recipients. The cellular and molecular mechanisms that underlie chronic rejection are not known, and their discovery can form the basis of clinical intervention. Several investigators have suggested that the development of chronic rejection in solid organ transplants is dependent on help mediated by CD4(+) lymphocytes. Importantly, the mechanism through which help is provided has not been fully delineated in transplant rejection. Using a murine heterotopic heart transplant model without immunosuppression, this study defines the functional role of CD4(+) lymphocytes in chronic rejection. In an MHC class II-mismatched model, we demonstrate that chronic rejection was absolutely contingent on the presence of CD4(+) lymphocytes. Importantly, here we report that signaling through CD40 can replace the requirement of CD4(+) lymphocytes, demonstrated by the development of chronic rejection in CD4 knockout recipients treated with a CD40-activating mAb (FGK45). The return of rejection appears to be a CD8(+) lymphocyte-dependent process, noted by the absence of rejection in FGK45-treated recombinase-activated gene knockout (CD4(+) and CD8(+) lymphocyte-deficient) recipients. The CD40 signaling pathway works independently of B7-CD28 costimulation, as indicated by the development of severe chronic rejection in CD28 knockout recipients. Importantly, this study provides evidence that CD40 ligand-targeted therapies may prevent chronic rejection only in strain combinations where CD4(+) lymphocyte help is absolutely required.     

Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell-cell contacts of polarized epithelial cells

Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins. Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion. In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins. A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis. The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones. Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells. Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells.     

Protein kinase C isozymes and the regulation of diverse cell responses

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

Structure, location, and lipid perturbations of melittin at the membrane interface

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.     

Factors limiting fish populations in the Loch Fleet system, an acidic drainage system in south-west Scotland

Loch Fleet is an oligotrophic upland lake in Galloway, south-west Scotland. It once supported a brown trout, Salmo trutta L., sport fishery with low annual catches (< 150 fish year ^?1) but catches declined markedly after 1950 and no fish were caught after 1975. Diatom records for the lake sediments indicate acute acidification since 1975, pH changing from c. 5–8 to 4–6. In 1984 a project was set up at Loch Fleet to investigate techniques of acidity mitigation, with a view to restoring fisheries in this and similarly affected waters. An underlying assumption of the project was that fish had been lost as a direct result of acidity and associated factors. Studies were therefore undertaken during a 2-year baseline period (1984–1986) to validate this assumption. Fish surveys using a variety of techniques (gill-netting, trapping, electrofishing) confirmed that trout were absent from the Loch and its afferent streams, and also from its main outlet stream, the Little Water of Fleet, for a distance of 7 km downstream. Trout were present below this point but are prevented from passing upstream by a 5-m waterfall. Eels, Anguilla anguilla L., were present throughout the Little Water of Fleet, though not in the Loch itself. Population densities of both species were low, with less than 7 eels and 5 trout per 100 m². Survival studies using brown trout ova and yolk-sac fry indicated that conditions in the Loch and its afferent streams were acutely toxic to these stages as a result of the low pH (pH 4.5), low calcium (I mg l^?1) and high aluminium concentrations (200 μg 1 ^?1 total Al, 60 μg 1 ^?1 inorganic monomeric Al). Trout fingerlings could survive these conditions in short-term tests (9 days) but, in chronic exposure tests lasting up to 180 days carried out in situ in streams adjoining the loch, no fish survived this period. This toxicity was eliminated in experiments where pH was raised to 5.4 by KOH addition. It is concluded that the loss of the brown trout fishery at Loch Fleet occurred as a direct result of acidity and related factors, probably acting in the first instance on the sensitive intra-gravel ova and yolk-sac fry stages, leading to recruitment failure.