Control mechanisms governing the infectivity of Chlamydia trachomatis for hela cells: modulation by cyclic nucleotides, prostaglandins and calcium

Chlamydia trachomatis causes common infections of the eyes and genital tract in man. The mechanism by which this obligate intracellular bacterium is taken into epithelial cells is unclear. The results described here support the concept that chlamydial infections of HeLa cells is under bidirectional cyclic nucleotide control, with guanosine 3':5'-cyclic monophosphate (cGMP) acting as a stimulator, and adenosine 3':5'-cyclic monophosphate (cAMP) as an inhibitor. Treatment of the HeLa cells with the divalent cation ionophore A23187, with carbamoylcholine, or with prostaglandins known to increase the concentration of endogenous cGMP, also increased host cell susceptibility to chlamydial infection. Cyclic GMP was only effective if added at or before chlamydial inoculation, suggesting that its main effect was on chlamydial uptake. The stimulatory effect of cGMP, but nt antagonism, by cAMP, was abolished if the cells were first treated with any of four different inhibitors of prostaglandin synthesis, suggesting a critical role for endogenous prostaglandin synthesis. Centrifugation of chlamydiae on to host cells was followed by a rapid increase in the mobility of Ca2+ across the cell membrane. The interrelationships of these observations and the possibility that chlamydiae and other intracellular pathogens might evoke alterations in host cell prostaglandin and cyclic nucleotide concentrations to aid their own uptake are discussed.     

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

Evidence of a role for calmodulin in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum

The effect of calmodulin and calmodulin inhibitors on the "Ca2+ release channel" of "heavy" skeletal muscle sarcoplasmic reticulum (SR) vesicles was investigated. SR vesicles were passively loaded with 45Ca2+ in the presence of calmodulin and its inhibitors, followed by measurement of 45Ca2+ release rates by means of a rapid-quench-Millipore filtration method. Calmodulin at a concentration of 2-10 microM reduced 45Ca2+ efflux rates from passively loaded vesicles by a factor of 2-3 in media containing 10(-6)-10(-3) M Ca2+. At 10(-9) M Ca2+, calmodulin was without effect. 45Ca2+ release rates were varied 1000-fold (k1 approximately equal to 0.1-100 s-1) by using 10(-5) M Ca2+ with either Mg2+ or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) in the release medium. In all instances, a similar 2-3-fold reduction in release rates was observed. At 10(-5) M Ca2+, 45Ca2+ release was half-maximally inhibited by about 2 X 10(-7) M calmodulin, and this inhibition was reversible. Heavy SR vesicle fractions contained 0.1-02 micrograms of endogenous calmodulin/mg of vesicle protein. However, the calmodulin inhibitors trifluoperazine, calmidazolium, and compound 48/80 were without significant effect on 45Ca2+ release at concentrations which inhibit calmodulin-mediated reactions in other systems. Studies with actively loaded vesicles also suggested that heavy SR vesicles contain a Ca2+ permeation system that is inhibited by calmodulin.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.     

Regulation by calmodulin of the calcium affinity of the calcium-transport ATPase in human erythrocytes

The Ca2+ affinity of (Mg2+ + Ca2+)-ATPase in human red blood cells is regulated by a number of intracellular factors, including the association of the enzyme with the cytosolic Ca2+ binding protein, calmodulin. Ghosts prepared by hypotonic lysis in the presence of 0.1 mM CaCl2, or by a gradual stepwise hemolysis procedure, contain an EDTA-extractable protein whose effects are mimicked by calmodulin, whereas ghosts prepared by extensive washes in the absence of added CaCl2 lack calmodulin and contain only a high molecular weight heat stable activator. Purified calmodulin from human red cells or bovine brain shifts the apparent Ca2+ affinity of (Mg2+ + Ca2+)-ATPase activity in extensively washed ghosts to a high Ca2+ affinity state. The shift was most apparent in ghosts in which the Ca2+ affinity was decreased by EDTA treatment. Calmodulin increased the velocity of (Mg2+ + Ca2+)-ATPase in the EDTA-treated ghosts about 36-fold at a low (1.4 microM) Ca2+ concentration, compared with 6-fold before EDTA treatment. The maximum shift in apparent Ca2+ affinity occurred only in the presence of saturating concentrations of calmodulin. It is concluded that red cell calmodulin confers to the Ca2+ transport ATPase the ability to increase its apparent Ca2+ affinity, as well as its maximum velocity, in response to increases in intracellular Ca2+.     

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

Acceleration of trophoblast differentiation by heparin-binding EGF-like growth factor is dependent on the stage-specific activation of calcium influx by ErbB receptors in developing mouse blastocysts

Heparin-binding EGF-like growth factor (HB-EGF) is expressed in the mouse endometrial epithelium during implantation exclusively at sites apposed to embryos and accelerates the development of cultured blastocysts, suggesting that it may regulate peri-implantation development in utero. We have examined the influence of HB-EGF on mouse trophoblast differentiation in vitro and the associated intracellular signaling pathways. HB-EGF both induced intracellular Ca2+ signaling and accelerated trophoblast development to an adhesion-competent stage, but only late on gestation day 4 after ErbB4, a receptor for HB-EGF, translocated from the cytoplasm to the apical surface of trophoblast cells. The acceleration of blastocyst differentiation by HB-EGF was attenuated after inhibition of protein tyrosine kinase activity or removal of surface heparan sulfate, as expected. Chelation of intracellular Ca2+ blocked the ability of HB-EGF to accelerate development, as did inhibitors of protein kinase C or calmodulin. The absence of any effect by a phospholipase C inhibitor and the requirement for extracellular Ca2+ suggested that the accrued free cytoplasmic Ca2+ did not originate from inositol phosphate-sensitive intracellular stores, but through Ca2+ influx. Indeed, N-type Ca2+ channel blockers specifically inhibited the ability of HB-EGF to both induce Ca2+ signaling and accelerate trophoblast development. We conclude that HB-EGF accelerates the differentiation of trophoblast cells to an adhesion-competent stage by inducing Ca2+ influx, which activates calmodulin and protein kinase C. An upstream role for ErbB4 in this pathway is implicated by the timing of its translocation to the trophoblast surface.     

Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.     

The role of calmodulin on Ca2+ -dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KC1 from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KC1 fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.     

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.     

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition.     

Regulation of Plant Defense Response to Fungal Pathogens: Two Types of Protein Kinases in the Reversible Phosphorylation of the Host Plasma Membrane H+-ATPase

The role of reversible phosphorylation of the host plasma membrane H+-ATPase in signal transduction during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum was investigated. Tomato cells (with the Cf-5 resistance gene) or isolated plasma membranes from Cf-5 cells treated with elicitor preparations from race 2.3 or 4 of C. fulvum (containing the avr5 gene product) showed a marked dephosphorylation of plasma membrane H+-ATPase. Similar treatment with elicitor preparations from races 5 and 2.4.5.9.11 (lacking the avr5 gene product) showed no change in dephosphorylation. Elicitor (race 4) treatment of cells, but not of isolated plasma membranes, for 2 hr resulted in rephosphorylation of the ATPase via Ca2+-dependent protein kinases. The initial (first hour) rephosphorylation was enhanced by protein kinase C (PKC) activators and was prevented by PKC inhibitors. Activity of a second kinase appeared after 1 hr and was responsible for the continuing phosphorylation of the H+-ATPase. This latter Ca2+-dependent kinase was inhibited by a calmodulin (CaM) antagonist and by an inhibitor of Ca2+/CaM-dependent protein kinase II. The activation of the Ca2+/CaM-dependent protein kinase depended on the prior activation of the PKC-like kinase.     

Endothelins stimulate aldosterone secretion from dispersed rat adrenal zona glomerulosa cells, acting through ETB receptors coupled with the phospholipase C-dependent signaling pathway

Compelling evidence indicates that endothelins (ETs) stimulates aldosterone secretion from rat zona glomerulosa (ZG) cells, acting through the ETB receptor subtype. We have investigated the mechanisms transducing the aldosterone secretagogue signal elicited by the pure activation of ETB receptors. Aldosterone response of dispersed rat ZG cells to the selective ETB-receptor agonist BQ-3020 was not affected by inhibitors of adenylate cyclase/protein kinase (PK)A, tyrosine kinase-, mitogen-activated PK-, cyclooxygenase- and lipoxygenase-dependent pathways. In contrast, the inhibitor of phospholipase C (PLC) U-73122 abrogated, and the inhibitors of PKC, phosphatidylinositol trisphosphate (IP(3))-kinase and calmodulin (calphostin-C, wortmannin and W-7, respectively) partially prevented aldosterone response to BQ-3020. When added together, calphostin-C and wortmannin or W-7 abolished the secretagogue effect of BQ-3020. BQ-3020 elicited a marked increase in the intracellular Ca2+ concentration ([Ca2+]i) in dispersed rat ZG cells, and the effect was abolished by the Ca(2+)-release inhibitor dantrolene. The Ca2+ channel blocker nifedipine affected neither aldosterone nor Ca2+ response to BQ-3020. Collectively, our findings suggest that (1) ETs stimulate aldosterone secretion from rat ZG cells through the activation of PLC-coupled ETB receptors; (2) PLC stimulation leads to the activation of PKC and to the rise in [Ca2+]i with the ensuing activation of calmodulin; and (3) the increase in [Ca2+] is exclusively dependent on the stimulation of IP(3)-dependent Ca2+ release from intracellular stores.     

Modulation of active Ca2+ uptake by the islet-cell endoplasmic reticulum.

The possible effects of calmodulin and cyclic AMP on active Ca2+ uptake by the islet-cell endoplasmic reticulum were investigated. Neither calmodulin nor cyclic AMP affected the rate of active Ca2+ uptake, or the steady-state filling capacity of the endoplasmic reticulum when measured in the absence of oxalate. Consistent with these results, calmodulin did not activate the Ca2+-stimulated ATPase activity associated with this cell fraction. During the course of these experiments., it was unexpectedly discovered that the rate of Ca2+ uptake, as well as the steady-state Ca2+ filling capacity of the endoplasmic reticulum, were markedly increased by unidentified factor(s) in the cytosol. This effect could be demonstrated by reconstitution of the membranes in cytosol, or by direct addition of fresh or dialysed cytosol to the Ca2+ uptake assays. The degree of activation by the cytosol indicates that the endoplasmic reticulum may play a prominent role in controlling beta-cell Ca2+ concentrations and that the unidentified activator(s) present in the cytosol may be involved in regulation of this function.     

The multifaceted role of oestrogen in enhancing Chlamydia trachomatis infection in polarized human endometrial epithelial cells

The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.     

Insulin release and protein phosphorylation: possible role of calmodulin

Both Ca2+ and cyclic AMP (cAMP) are implicated in the regulation of insulin release in the pancreatic beta cell. In hamster insulinoma cells used in our laboratory to study the mechanism of insulin release, Ca2+ and cAMP trigger secretion independently. Concomitant with stimulation of the secretory apparatus both cAMP and Ca2+ promote phosphorylation of distinct insulinoma cell proteins. Calmodulin may be involved in the stimulation of insulin release and protein phosphorylation induced by Ca2+ influx. The Ca2+-dependent protein kinase of the insulinoma cell is activated by exogenous calmodulin and blocked by trifluoperazine, and inhibitor of calmodulin action. This drug also inhibits glucose-induced insulin release in pancreatic islets. In insulinoma cells trifluoperazine blocks Ca2+ influx-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated secretion. Inhibition of Ca2+ influx-mediated insulin release and protein phosphorylation occurs with nearly identical dose dependence. Inasmuch as trifluoperazine affects voltage-dependent Ca2+ uptake in insulinoma cells, an involvement of calmodulin cannot be directly inferred. The evidence suggests that protein phosphorylation may be involved in the activation of the secretory apparatus by both cAMP and Ca2+. It is proposed that stimulation of insulin release by cAMP and Ca2+ is mediated by cAMP-dependent protein kinase and calmodulin-dependent protein kinase, respectively.