Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.     

Metabolic effects of trifluoperazine in the liver and the influence of calcium

The effects of trifluoperazine on hepatic cell metabolism were investigated using isolated perfused rat liver. The following effects of trifluoperazine were found: (1) trifluoperazine inhibited oxygen uptake, the site of action being the mitochondria. Half-maximal inhibition occurred at concentrations around 50 microM; with 100 microM trifluoperazine the effect was already maximal. When Ca2+ was withdrawn from the perfusion medium and the intracellular Ca2+ pools were exhausted, the inhibitory action on respiration was no longer observable. The reintroduction of Ca2+ restored inhibition. (2) Glycogenolysis and glycolysis were not significantly affected during the infusion of trifluoperazine. After stopping trifluoperazine infusion, however, glycogenolysis (glucose release) experienced a transitory stimulation. (3) Gluconeogenesis from lactate as the carbon source was inhibited by trifluoperazine. This inhibition was approximately proportional to the inhibition of oxygen uptake. Withdrawal of Ca2+ diminished, but it did not eliminate, inhibition of gluconeogenesis. (4) Ketogenesis was also inhibited in parallel with the inhibition of oxygen uptake. Withdrawal of Ca2+ from the perfusion fluid also abolished this action. (5) The effects of trifluoperazine were reverted very slowly when its infusion was stopped. The recovery of oxygen uptake at 50 min after cessation of the infusion was only 30%. Uptake of the substance was very fast. Absence of Ca2+ did not affect uptake. It was concluded that inhibition of mitochondrial energy metabolism is one of the most prominent effects of trifluoperazine in the liver. The fact that this inhibition depends on Ca2+ is unique.     

Enhancement of vascular action of arginine vasopressin by diminished extracellular sodium concentration

The present study was undertaken to examine the effects of diminished extracellular sodium concentration on the vascular action of arginine vasopressin (AVP) in cultured rat vascular smooth muscle cells (VSMC). The preincubation of cells with the 110 mM extracellular Na+ ([Na+]e) solution supplemented with 30 mM choline chloride for 60 minutes enhanced the effect of AVP- (1 x 10(-8) M) induced VSMC contraction. The treatment of 110 mM [Na+]e solution also enhanced the cellular contractile response to the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetyl-glycerol. Furthermore, preincubation with the 110 mM [Na+]e solution also potentiated the effect of 1 x 10(-8) M AVP, but not 1 x 10(-6) M, to increase the cytosolic-free Ca2+ ([Ca2+]i) concentration. The 110 mM [Na+]e media decreased the basal intracellular Na+ concentration and increased intracellular 45Ca2+ accumulation, basal [Ca2+]i and AVP-produced 45Ca2+ efflux. These effects of 110 mM [Na+]e solution to enhance the vascular action of AVP were abolished by using Ca(2+)-free 110 mM [Na+]e solution during the preincubation period. The preincubation with the 110 mM [Na+]e solution did not change either the Kd and Bmax of AVP V1 receptor of VSMC or the AVP-induced production of inositol 1,4,5-trisphosphate. The present in vitro results therefore indicate that the diminished extracellular fluid sodium concentration within a range observed in clinical hyponatremic states enhances the vascular action of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electronmicroscopic study of human herpesvirus 6-infected human T cell lines superinfected with human immunodeficiency virus type 1

Melatonin release by chick cultured pineal cells increases during the dark periods and decreases during the light periods under light-dark cycles, and this rhythmic secretion is maintained under constant conditions with a period of almost 24 hr. The mechanisms by which the circadian oscillator drives the melatonin rhythm under constant conditions have not been elucidated enough. We examined the possibility that cyclic AMP-dependent protein kinase A is involved in the subjective nocturnal increase in melatonin release by chick pineal cells cultured under constant darkness. The subjective nocturnal increase of melatonin release was suppressed dose dependently by H8 (protein kinase inhibitor) and H89 (specific protein kinase A inhibitor), but not by calphostin C (specific protein kinase C inhibitor) in static cell cultures. In a cell perfusion experiment, 9 hr pulses of H8 and H89 starting at ZT 9 (CT 11.2) hr suppressed the subjective nocturnal increase in melatonin rhythm in dose-dependent manner without causing a phase shift. An intracellular Ca2+ chelator, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), and extracellular Ca2+ chelators, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetrapotassium salt hydrate (BAPTA) and ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), suppressed both the subjective nocturnal increases in melatonin release and cAMP levels dose dependently. This direct evidence strongly supports the hypothesis that cAMP-dependent protein kinase A may be involved in the subjective nocturnal increase in melatonin release by chick pineal cells and that intracellular Ca2+ plays an important role in pineal adenylate cyclase activation.     

Anti-fungal and cytokine producing activities of CD8 + T lymphocytes from HIV-1 infected individuals

Lymphokine activated killer (LAK) cells are capable of killing not only malignant cells but also hyphal form of Candida albicans in vitro. When peripheral blood mononuclear cells (PBMC) from normal healthy donors were cultured for 72-96 hrs with 1,500 international unit (IU)/ml interleukin-2 (IL-2), marked LAK activity was induced. However, even prior to IL-2 activation, PBMC isolated from some normal subjects and those from almost all individuals who are infected by human immunodeficiency virus type 1 (HIV-1) exhibited significant levels of anti-fungal activity. Such pre-activation ("in situ") antifungal activity of PBMC decreased during the initial 48 hrs of IL-2 activation. PBMC from HIV-1 seropositive subjects showed higher levels of "in situ" anti-fungal activity than normal PBMC did. After a decline of "in situ" activity during the initial 48 hours, LAK activity gradually increased and reached near maximal levels by day 4 and remained more or less constant until day 6. No significant difference was observed between the LAK activity of normal and HIV-1(+) PBMCs on days 4-6. In IL-2 activated normal and HIV-1(+) PBMC cultures, both CD4 and CD8 T cells produced IL-2, INF-gamma as well as TNF-alpha. Production of IL-2 by both CD4 and CD8 T cells was suppressed in HIV-1(+) PBMC cultures, but no significant suppression of INF-gamma production was noted. Meanwhile, TNF-alpha production by CD4 was very much suppressed but no significant changes in TNF-alpha production by CD8 T cells was noted in HIV-1(+) PBMC cultures.     

Mechanisms by which thionin induces susceptibility of S49 cell membranes to extracellular phospholipase A2

Whereas cells normally resist attack by PLA2, they become susceptible under certain pathological conditions. To ascertain the regulatory mechanisms that induce cellular susceptibility to PLA2, the effect of thionin on S49 cells was examined in the presence of PLA2. Thionin alone was unable to evoke hydrolysis of the lipid bilayer. Likewise, the addition of PLA2 alone caused production of only a minimal amount of free fatty acid. However, thionin and PLA2 together resulted in significant hydrolysis of the cell membrane. Thionin caused perturbation of the bilayer structure as suggested by the changes in the emission spectra of laurdan and the permeability of the membrane to propidium iodide. These changes correlated quantitatively with the susceptibility of the lipid bilayer to PLA2. Furthermore, thionin induced a modest increase in intracellular Ca2+. The source of this Ca2+ was the extracellular fluid since EDTA in the extracellular medium inhibited the Ca2+ influx. Moreover, cobalt chloride, a universal Ca2+ channel blocker, prevented the rise in intracellular Ca2+, the uptake of propidium iodide, and the susceptibility to PLA2 induced by thionin. In contrast, the changes in the laurdan emission caused by the thionin were not affected by the cobalt. Furthermore, incubation of the cells with the calcium ionophore A23187 also caused the cells to become susceptible to PLA2. We hypothesize that thionin causes S49 cell membranes to become susceptible to PLA2 by a Ca2+-dependent perturbation of the bilayer structure.     

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.     

Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

Fluid flow modulates vascular endothelial cytosolic calcium responses to adenine nucleotides

OBJECTIVE: To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. METHODS: Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca(2+)-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+]i) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. RESULTS: Continuous perfusion with ATP (0.3-3.0 microM) or ADP (0.1-1.0 microM) evoked repetitive oscillations in [Ca2+]i in individual BAEC. The frequency of the [Ca2+]i oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]i oscillations. In the presence of nonhydrolyzable nucleotide analog, ATP gamma S or ADP beta S, application of flow resulted in similar shear-dependent [Ca2+]i oscillations, suggesting that flow modulation of the [Ca2+]i response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. CONCLUSIONS: These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Reproductive health law: where next, after Cairo and Beijing?

OBJECTIVE: Diabetes selectively injures receptor-mediated endothelium-dependent relaxation. In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells. METHODS: [Ca2+i] was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production. RESULTS: After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell [Ca2+i] in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered. When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in [Ca2+i] signal was observed after exposure to 35 mM glucose for a similar time period. Defective [Ca2+i] signaling was also seen in cells using MEM medium. [Ca2+i] signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure. The defect in [Ca2+i] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake. Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca2+i] in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure. CONCLUSIONS: Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.     

Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.     

Differential role of calcium in tumour necrosis factor-mediated apoptosis and secretion of granulocyte-macrophage colony-stimulating factor in a T cell hybridoma

The authors investigated the dependence on extracellular and intracellular free Ca2+ in the induction of apoptosis and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by tumour necrosis factor (TNF) in a rat/mouse T cell hybridoma PC60 R55/R75, using the Ca2+ chelators EGTA and BAPTA/AM, respectively. TNF-induced apoptosis still occurred in the absence of free Ca2+, while GM-CSF production required the continuous presence of Ca2+. The latter was also true for GM-CSF production driven by interleukin 1 (IL-1). The dependence on Ca2+ in the induction of GM-CSF, but not of apoptosis, was further confirmed by the inhibition of TNF- or IL-1-induced cytokine production by cyclosporin A or FK506, drugs that block the Ca2+/calmodulin-dependent protein Ser/Thr phosphatase calcineurin. This differential requirement for Ca2+ illustrates the partial functional redundancy between TNF and IL-1, showing the activation of cytokine gene expression through a Ca(2+)-dependent activation of calcineurin, and a Ca(2+)-independent activation of apoptosis, exerted solely by TNF.     

Demonstration of human T lymphotropic virus type I (HTLV-I) tax-specific CD8+ lymphocytes directly in peripheral blood of HTLV-I-associated myelopathy/tropical spastic paraparesis patients by intracellular cytokine detection

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-I infection and has been associated with elevated levels of several proinflammatory cytokines in both serum and cerebrospinal fluid. It is unknown what kind of cells secrete these cytokines and if HTLV-I Ags are associated with this phenomenon. Here, we investigated the expression of cytokines in PBL from eight HAM/TSP patients, nine HTLV-I-infected asymptomatic carriers, and seven healthy controls by flow cytometry combined with intracellular cytokine staining. PBL were cultured with brefeldin A without mitogen and IL-2 for 14 h. Under these conditions, CD8+ cells produced proinflammatory cytokines including IFN-gamma, TNF-alpha, and IL-2, which were significantly elevated in HAM/TSP patients. The proportion of CD8+ cells producing IFN-gamma in HAM/TSP patients, asymptomatic carriers, and healthy controls were, on average, 4.9, 0.4, and 0.3%, respectively. IFN-gamma production by these CD8+ cells was suppressed by anti-HLA-class I Ab. Purified CD8+ cells from an HLA-A2 HAM/TSP patient produced IFN-gamma by cocultivation with autologous CD4 cells, the main reservoir of HTLV-I in vivo, or allogenic HLA-A2+ B cells pulsed with a known immunodominant HTLV-I tax peptide. These data suggest that high levels of circulating HTLV-I-specific CD8+ T lymphocytes have the potential to produce proinflammatory cytokines and may promote inflammatory responses to HTLV-I in HAM/TSP patients.     

Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos

To determine the role of intracellular Ca2+ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca2+, blocking of Ca2+ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca2+ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca2+ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell-cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca2+ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca2+. Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca2+ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca2+ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.     

Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.