Effect of angiotensin II and ethanol on the expression of connexin 43 in WB rat liver epithelial cells.

The turnover of connexin 43 (Cx43) is very rapid in many cells and involves both the lysosomal and proteasomal protease pathways. Here we show that Ca(2+)-mobilizing agonists such as angiotensin II (Ang II) can up-regulate the expression of Cx43 in WB rat liver epithelial cells. Vasopressin had the same effect in A7R5 smooth-muscle cells. The effect of Ang II was not prevented by pretreatment with proteasomal or lysosomal inhibitors and was associated with an enhanced biosynthesis of Cx43 as measured by metabolic labelling experiments. The accumulation of Cx43 occurred in intracellular compartments and at the cell surface, as determined by confocal immunofluorescence studies and by immunoblotting of fractions soluble and insoluble in Triton X-100. Chronic treatment of WB cells with ethanol inhibited Cx43 expression; this was associated with decreased biosynthesis of Cx43. Neither treatment with Ang II nor treatment with ethanol altered the levels of Cx43 mRNA. Incubation of WB cells with Ang II did not alter gap-junctional communication as judged by a dye-coupling assay. However, treatment with ethanol markedly decreased gap-junctional communication and this effect was diminished in Ang-II-treated cells, demonstrating that gap-junctional communication is linked to the level of Cx43 expression. We conclude that Cx43 biosynthesis is regulated by Ca(2+)-mobilizing agonists and ethanol in WB cells. The changes in Cx43 expression might have a role in modifying the conduction of metabolites and second messengers between cells.

Factors determining the composition of inositol trisphosphate receptor hetero-oligomers expressed in COS cells.

COS-7 cells were transiently transfected with type I and type III myo-inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms to study the processes underlying assembly and oligomerization of these tetrameric proteins. A FLAG epitope was engineered on to the N terminus of the type III IP(3)R to distinguish the transfected from the endogenous isoform. This was not necessary for the type I IP(3)R, since the endogenous levels of this isoform were extremely low. Based on sucrose gradient analysis, the transfected type I or FLAG-type III IP(3)Rs assembled into tetramers. Confocal immunofluorescence experiments confirmed that the constructs were primarily targeted to the endoplasmic reticulum. Recombinant type I IP(3)R expressed in COS cells over a 48-h period showed a negligible capacity to form hetero-oligomers with endogenous type III IP(3)Rs, based upon co-immunoprecipitation assays. However, substantial formation of hetero-oligomers was observed between recombinant receptors when the cells were simultaneously transfected with type I and FLAG-type III IP(3)Rs. Co-immunoprecipitation experiments using lysates from metabolically labeled cells allowed the quantitation of homo- and hetero-oligomers in cells transfected with different ratios of type I and FLAG-type III IP(3)R DNA. These studies show that the relative expression level of the two isoforms influences the fraction of hetero-oligomers formed. However, the proportion of hetero-oligomers formed were less than predicted by a binomial model in which the association of subunits is assumed to be random. In doubly transfected cells, the early kinetics of (35)S label incorporation into homotetramers showed a lag period corresponding to the time taken to synthesize a full-length receptor. However, hetero-oligomers were synthesized with a longer lag period, suggesting that there may be kinetic constraints that favor homo-oligomers over hetero-oligomers.

Biosynthesis of inositol trisphosphate receptors: selective association with the molecular chaperone calnexin.

A prominent labelled polypeptide having the same mobility as type-I inositol trisphosphate receptor (IP(3)R) was immunoprecipitated from WB-cell lysates by antibodies to calnexin, an ER integral membrane chaperone. The identity of this polypeptide was confirmed by re-immunoprecipitation of the radioactive polypeptides released from calnexin-antibody immunoprecipitates with type-I IP(3)R antibody. The interaction of calnexin with newly synthesized type-I IP(3)R was transient and inhibited by treatment of the cells with dithiothreitol or the glucosidase inhibitor N-methyldeoxynojirimicin. In similar experiments, there was no evidence for the binding of type-I IP(3)R to calreticulin, an ER luminal chaperone. Calnexin (but not calreticulin) associated with newly synthesized FLAG (DYKDDDDK epitope)-tagged type-III IP(3)R expressed in COS-7 cells. In order to further define the mechanism of interaction of nascent IP(3)R with chaperones, we have utilized an in vitro rabbit reticulocyte translation assay programmed with RNA templates encoding the six putative transmembrane (TM) domains of IP(3)Rs. In accordance with the known preference of calnexin for monoglucosylated oligosaccharide chains, calnexin antibody preferentially immunoprecipitated a proportion of glycosylated type-I translation product. Addition of glucosidase inhibitors prevented the association of calnexin with in vitro translated type-I TM construct. Using truncated RNA templates we found that calnexin did not associate with the first four TM domains but retained affinity for the construct encoding TM domains 5 and 6, which contains the glycosylation sites. We propose that calnexin is a key chaperone involved in the folding, assembly and oligomerization of newly synthesized IP(3) receptors in the ER.

Effect of chronic ethanol exposure on inositol trisphosphate receptors in WB rat liver epithelial cells.

BACKGROUND: Enhanced agonist-induced Ca2+ release has been reported in hepatocytes isolated from ethanol-fed rats. Because myo-inositol 1,4,5-trisphosphate receptors (IP3Rs) are involved in the mobilization of Ca2+, we examined the effects of chronic ethanol treatment on IP3R function and levels of IP3R protein by using WB rat liver epithelial cells. METHODS: WB cells were treated with ethanol (50-150 mM) for 24 to 48 hr and were loaded with Fura-2 to measure agonist-induced Ca2+ mobilization or saponin permeabilized to measure myo-inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. IP3 levels were measured in [3H]-inositol labeled cells. Levels of IP3R protein were quantitated by immunoblotting with antibodies to IP3R isoforms. Lysosomal and proteasomal peptidase activities were assayed in cytosol and membrane fractions using specific fluorogenic peptide substrates. RESULTS: Ethanol treatment enhanced Ca2+ mobilization in response to angiotensin II, vasopressin, and bradykinin. This effect was not due to an increased production of IP3. Chronic ethanol treatment stimulated the mobilization of Ca2+ from saponin-permeabilized cells in response to subsaturating doses of IP3 and increased the basal levels of both type I and type III IP3Rs by 1.8-fold and 1.6-fold, respectively. Ethanol treatment did not prevent angiotensin II-induced IP3R down-regulation or alter lysosomal cathepsin B activity or the trypsin-like and peptidylglutamyl peptidase activities of the proteasome. However, chronic ethanol exposure resulted in a 60% and 41% inhibition of the chymotrypsin-like activity of the proteasome in cytosol and microsomal membranes, respectively. CONCLUSION: We propose that the enhanced agonist-mediated Ca2+ mobilization observed in chronic ethanol-treated WB liver epithelial cells results from increased IP3R expression caused by an inhibition of IP3R degradation pathways by ethanol.

Transforming growth factor-beta1 inhibits type I inositol 1,4,5-trisphosphate receptor expression and enhances its phosphorylation in mesangial cells.

A potentially important cross-talk characteristic of transforming growth factor-beta (TGF-beta) is to inhibit platelet-derived growth factor-induced intracellular calcium rise (Baffy, G., Sharma, K., Shi, W., Ziyadeh, F. N., and Williamson, J. R. (1995) Biochem. Biophys. Res. Commun. 210, 378-383) in murine mesangial cells. The present study examined the possible basis for this effect by evaluating the regulation of the type I inositol 1,4,5-trisphosphate receptor (IP3R) by TGF-beta. TGF-beta1 down-regulates IP3R protein expression by >90% with maximal and half-maximal effects after 8 and 2 h, respectively. TGF-beta1 also decreased IP3R mRNA expression by 59% after 1 h. Phosphorylation of the IP3R was also demonstrated as early as 15 min after TGF-beta1 exposure. Back phosphorylation assays of IP3R from TGF-beta1-treated mesangial cells with protein kinase A (PKA), indicated that TGF-beta1-induced phosphorylation of the IP3R occurs at similar sites as for PKA. In vitro kinase assays using the known IP3R peptide substrates for PKA, RPSGRRESLTSFGNP and ARRDSVLAAS, demonstrated that TGF-beta1 induces phosphorylation of both peptides (158 and 123% of control values, respectively). TGF-beta1-induced phosphorylation was prevented by the addition of the PKA inhibitor peptide in the in vitro kinase assay. It is proposed that TGF-beta-mediated effects on the IP3R may be an important characteristic of its ability to modulate the response of cells to factors that employ IP3R-mediated calcium release.

Angiotensin II-induced down-regulation of inositol trisphosphate receptors in WB rat liver epithelial cells. Evidence for involvement of the proteasome pathway.

Chronic stimulation of WB rat liver epithelial cells by angiotensin II (Ang II) resulted in the down-regulation of both type I and type III myo-inositol 1,4,5-trisphosphate receptors (IP3Rs). Stimulation with vasopressin, bradykinin, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate was without effect. Ang II-induced down-regulation of IP3Rs could be detected within 2 h and resulted in an inhibition of IP3-induced Ca2+ release from permeabilized cells. IP3R down-regulation was reversible, and both homo- and heterooligomers of IP3Rs were equally susceptible to Ang II-induced degradation. Chloroquine and NH4Cl increased the basal levels of IP3Rs by 2-fold, suggesting that the basal turnover of IP3Rs occurs via a lysosomal pathway. However, Ang II-induced degradation of IP3R was not affected by these inhibitors, suggesting that stimulated degradation of IP3Rs occurs via a non-lysosomal pathway. The cysteine protease and proteasomal inhibitor N-acetyl-Leu-Leu-norleucinal completely prevented Ang II-mediated down-regulation of IP3Rs, whereas the structural analog N-acetyl-Leu-Leu-methioninal was without effect. Lactacystin, a highly specific proteasome inhibitor, also blocked Ang II-mediated IP3R degradation. Stimulation with Ang II increased the amount of IP3R immunoprecipitated by anti-ubiquitin antibodies. We conclude that Ang II-stimulated IP3R degradation involves enhanced ubiquitination of the protein and degradation by the proteasome pathway.

Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds.

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.

Localization and identification of Ca2+ATPases in highly purified human platelet plasma and intracellular membranes. Evidence that the monoclonal antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in human platelets.

The Ca2+ATPase activities of highly purified human platelet membranes prepared by high-voltage free-flow electrophoresis have been analysed by using [gamma-32P]ATP hydrolysis, recognition by antibodies and phosphoenzyme-complex formation. The Ca2+ATPase activity present in mixed membranes was found to be predominantly associated with intracellular membranes after subfractionation, with only a low level of activity associated with plasma membranes. The intracellular-membrane Ca2+ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca2+ATPase was not significantly affected, suggesting that the latter does not belong to the SERCA (sarco-endoplasmic-reticulum Ca2+ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca2+ATPase [Cheng, Magocsi, Cooper, Penniston and Borke (1993) Cell Physiol. Biochem. 4, 31-43] recognized two bands at 135 and 150 kDa in mixed membranes and plasma membranes, and the corresponding bands in red-blood-cell membranes, confirming the Ca2+ATPase to be of the PMCA (plasma-membrane Ca2+ATPase) type. No recognition of any band was detected in intracellular membranes. Identification of the intracellular-membrane Ca2+ATPase activity was carried out with polyclonal antibodies with known specificity towards SERCA 2b (S.2b) and SERCA 3 (N89), and a monoclonal antibody, PL/IM 430, raised against platelet intracellular membranes. All of these antibodies recognized the 100 kDa Ca2+ATPase in mixed membranes and intracellular membranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and antiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca2+ATPase recognized by PL/IM 430 was immunoprecipitated, and the immunoprecipitated protein was specifically recognized by the antiserum N89, but not by S.2b. Analysis of the phosphoenzyme-complex formation revealed potent phosphorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 430 and N89. These studies report the presence of a PMCA in a purified plasma-membrane fraction from human platelets, and that the antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in intracellular membranes.

A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.

An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.

Ca2+ release from platelet intracellular stores by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone: relationship to Ca2+ pools and relevance in platelet activation.

The effects of the Ca(2+)-ATPase inhibitors thapsigargin (Tg) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca(2+)-regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca(2+)-ATPase activities of platelet mixed membranes, without any effect on the basal Mg(2+)-ATPase activity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM). The effect of the two inhibitors on 45Ca2+ release from saponin-permeabilized platelets has also been characterized. 45Ca2+ uptake into non-mitochondrial intracellular stores occurs via an ATP-dependent mechanism, and if added at equilibrium the second messenger Ins(1,4,5)P3 releases 50% of the accumulated 45Ca2+. Maximally effective concentrations of Tg (1 microM) and tBuBHQ (50 microM) release 77% and 68% of the accumulated 45Ca2+. Addition of Ins(1,4,5)P3 together with either Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-sensitive Ca2+ pool was a subset of the pool that is sensitive to the Ca(2+)-ATPase inhibitors. Release of 45Ca2+ by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca2+ release, and Tg was found not to affect [32P]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool without affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Both Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cells already treated with Tg or tBuBHQ produced further elevation of Ca2+, indicating agonist utilization of a Ca(2+)-ATPase inhibitor-insensitive pool. In aggregation experiments Tg and tBuBHQ showed different functional effects. In indomethacin-treated cells Tg induces slow aggregation and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kinase C activator dioctanoylglycerol (DiC8). Tg also showed greater ability than tBuBHQ to release [3H]arachidonic acid (AA) from [3H]AA-labelled platelets. Additionally, in [32P]Pi-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light chain, a 27 kDa protein and the 45 kDa protein pleckstrin, but Tg showed a greater ability than tBuBHQ to cause phosphorylation of pleckstrin. These studies indicate that Tg and tBuBHQ are effective in releasing the Ins(1,4,5)P3-sensitive Ca2+ pool in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)