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PubMed2002

Pulmonary surfactant proteins A and D directly suppress CD3+/CD4+ cell function: evidence for two shared mechanisms.

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3(+)/CD4(+) lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-gamma when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca(2+) ](i)), but not thapsigargin-induced changes in [Ca(2+)](i). In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca(2+)](i).

Amino Acid Sequence↗
PubMed2000

Modulation of tumour necrosis factor production with desmuramyldipeptide analogues.

Some synthetic analogues of the immunomodulatory agent muramyl dipeptide (MDP), i.e. phthalimido- (LK-511, LK-413, LK-512, LK-423, LK-508), adamantyl- (LK-415, LK-517), 7-oxoalkyl-(LK-409) desmuramylpeptides were assessed for the tumour necrosis factor (TNF) inducing activity and the ability to modulate TNF production in in vitro phorbol 12-myristate 13-acetate (PMA) & ionomycin stimulated cultures of human peripheral blood mononuclear cells. A kinetic study over a 40-hour period indicated that desmuramyldipeptides were weak TNF inducers compared to romurtide, PMA & ionomycin or lipopolysaccharide. By contrast, they showed the potential to up- or down-regulate the production of TNF evoked by PMA & ionomycin, which was strongly dependent on the time of the stimulation. After 4h of stimulation, the TNF secretion was augmented by LK-508, LK-409 and LK-511, after 18 h by LK-409 and LK-423, and after 40 h by LK-423, LK-511, LK-415 and LK-512. However, LK-517 and LK-512 inhibited the secretion of TNF after the 18-h period.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.

Animals↗
PubMed1992

[Study of the possibility to abolish the action of immunosuppressive factors of tumor cells].

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Induction and regulation of casein kinase II during B lymphocyte activation.

This study evaluates the regulation of casein kinase II (CK II) activity in resting B cells induced to enter the cell cycle. The induction of B cell cycle progression PMA and ionomycin results in an oscillatory expression of CK II. This kinase activity is also elicited after direct physical interaction between B cells and activated, fixed Th cells, indicating that the increase seen in CK II activity is probably associated with the delivery of the competence-inducing signal to resting B cells. The selective inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, during PMA and ionomycin-induction of B cell cycle progression, inhibits the expression of CK II activity. The addition of polyamines to cytosolic preparations recovered from cells in which ODC is inhibited results in the appearance of CK II activity, showing that the ODC inhibitor does not directly inhibit the kinase. The treatment of B cells with cycloheximide results in the appearance of CK II activity within 15 min, and this induction is partially explainable by a cycloheximide-elicited increase in cellular levels of polyamines. The artificial elevation of cellular levels of cAMP simultaneous with the addition of PMA and ionomycin results in a 150 to 200% increase in detectable CK II levels, suggesting that the cAMP-dependent signaling cascade may participate during the early regulation of CK II. In contrast, the inhibition of protein kinase C does not adversely influence the early expression of CK II, while actually enhancing kinase activity by 18 h poststimulation.

Animals↗
PubMed1991

Association of various T cell-surface molecules with the cytoskeleton. Effect of cross-linking and activation.

The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.

Adult↗
PubMed1990

Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin.

FK-506 and the structurally related macrolide rapamycin (RAP) were investigated in comparison with cyclosporin A (CsA) for their immunosuppressive effects on murine T cells. All three agents suppressed the proliferation of splenic T cells triggered by lectins or antibodies to CD3 and Ly-6C. FK-506 or CsA also inhibited proliferation, IL-2 production, and IL-2R expression in splenic T cells activated with ionomycin + PMA. However, RAP minimally affected IL-2 production and IL-2R expression in these cells, although it reduced proliferation. Similarly, FK-506 and CsA, but not RAP, suppressed IL-2 production by activated DO.11.10 T hybridoma cells. In such a system, as well as in normal T cells stimulated with high ionomycin concentrations, FK-506 and CsA enhanced proliferation, indicating that they both abrogate negative signals associated with T cell activation. On the contrary, RAP diminished the autonomous proliferation of hybridoma cells, whereas FK-506 and CsA had little effect. The proliferative response induced in D10.G4 cells by IL-1 + ionomycin but not that induced by IL-1 + PMA was sensitive to inhibition by FK-506 and CsA. In contrast, RAP inhibited equally well both types of stimulation. Finally, T cell proliferation driven by IL-2 or IL-4 was found to be relatively resistant to FK-506 or CsA but sensitive to RAP. Altogether, these data demonstrate that FK-506 and CsA alter similar calcium-associated events of T cell activation and block T cell proliferation primarily by suppressing lymphokine production. RAP interferes with a different set of events and inhibits T cells by impairing their response to growth-promoting lymphokines.

Animals↗
PubMed1992

Protection of human umbilical vein endothelial cells by glycine and structurally similar amino acids against calcium and hydrogen peroxide-induced lethal cell injury.

Cultured human umbilical vein endothelial cells treated with either the calcium ionophore, ionomycin, or ionomycin plus cyanide-m-chlorophenylhydrazone had immediate severe depletion of adenosine triphosphate, (ATP) and increases of cytosolic free calcium (Caf) and then sustained lethal cell injury as manifested by release of lactate dehydrogenase and failure to exclude vital dyes within 15 minutes. Inclusion of glycine in the experimental medium prevented the enzyme leakage for at least 60 minutes without altering the ATP depletion or increases of Caf. The physiologic glycine concentration of 0.25 mmol/l gave 50% protection, and protection was complete at 1 mmol/l. Several other small neutral amino acids, L- and D-alanine, beta-alanine, 1-aminocyclopropane-1-carboxylate, alpha-aminoisobutyrate, and L-serine, had effects similar to glycine, but other amino acids and metabolic substrates did not. The endothelial cells were relatively resistant to damage from hydrogen peroxide, but sensitivity could be increased by preloading with Fe2+. In both non-loaded and Fe(2+)-loaded cells, hydrogen-peroxide-induced lactate dehydrogenase (LDH) release developing over 180 minutes was prevented by glycine in a fashion analogous to that seen with ionomycin damage. Mn2+ also partially protected against hydrogen peroxide injury but was not required for glycine's effects. These data demonstrate that striking modulatory effects of glycine and structurally similar amino acids that have previously been characterized in most detail using kidney tubule cells are strongly expressed in human umbilical vein endothelial cells and are involved in their response to Ca2+ and oxidant-mediated damage. These amino acid effects must be considered in the design of in vitro studies of endothelial cell injury and may contribute to endothelial cell pathophysiology in vivo.

Amino Acids↗
PubMed1992

Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol.

HepG2 cells were employed as model system to investigate potential relationships between early protein processing and Ca2+ storage by the endoplasmic reticulum. Ca2+ was required for glycoprotein processing and export by intact cells. The processing and export of alpha 1-antitrypsin and the secretion of complement factor 3, which are glycosylated proteins, were inhibited by the Ca2+ ionophore ionomycin whereas the export of albumin, a non-glycoprotein, was little affected. Ionomycin blocked processing of alpha 1-antitrypsin at the conversion from the high mannose to the complex glycosylated form without affecting ATP or GTP contents. Pre-existing inhibition of intracellular processing of alpha 1-antitrypsin by ionomycin was fully reversible upon removal of the ionophore with fatty acid-free bovine serum albumin. This reversal required Ca2+. After reversal the arrested form of alpha 1-antitrypsin was fully converted to the mature form and exported to the medium. Inhibitions of alpha 1-antitrypsin processing and complement factor 3 secretion by the metalloendoprotease antagonist Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) were strongest at low extracellular Ca2+ but were reduced or prevented by high extracellular Ca2+. Processing and secretion of alpha 1-antitrypsin were reduced upon incubation in low Ca2+ medium. Exposure to dithiothreitol reduced albumin export while affecting alpha 1-antitrypsin export minimally. Suppression of amino acid incorporation into total cellular proteins of HepG2 cells accompanied inhibitions of protein processing by agents depleting sequestered Ca2+ stores or by dithiothreitol. Putative control of rates of translational initiation by the endoplasmic reticulum through linkage to rates of early protein processing is discussed.

Amino Acids↗
PubMed1991

Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.

Animals↗
PubMed1991

Detection of hyperreactive T cells in multiple myeloma by multivalent cross-linking of the CD3/TCR complex.

Cellular immunity was investigated in 43 patients with multiple myeloma (MM) by assessing 3HTdR uptake induced by monocyte-dependent [CD3 monoclonal antibodies (MoAbs), phytohemagglutinin (PHA)] and monocyte-independent (CD2 MoAbs, ionomycin + phorbolester) stimulations. The former were evaluated in peripheral blood mononuclear cells (PBMNC) and purified T cells; the latter were evaluated in purified T-cell preparations only. MM showed significantly lower PBMNC responses to PHA (P less than .001), soluble OKT3 (CD3) (P = .01), and immobilized OKT3 MoAbs (P = .01). On purification of T cells, MM responses were still defective to soluble T11(2) + T11(3) (CD2) MoAbs (P = .004), phorbol myristate acetate (PMA) plus ionomycin (P less than .001), but significantly higher to plastic-immobilized OKT3 (P = .004). In some MM, 3HTdR uptake, interleukin-2 (IL-2) receptor (CD25) expression, and IL-2 production were as high on stimulation with plastic-immobilized OKT3 as that observed in normal subjects under optimal conditions (ie, plastic-immobilized OKT3 plus accessory signals). CD3 hyperreactivity correlated with the number of CD8+ HLA-DR+ cells in MM T-cell preparations. MM patients with more than 10% CD8+ HLA-DR+ cells had significantly higher responses to immobilized OKT3 (P less than .001), but lower responses to T11(2) plus T11(3) (P = .01), and PMA plus ionomycin (P = .03) than patients with less than 10% CD8+ HLA-DR+ cells. Phenotyping of CD45RA (naive) and CD45R0 (memory) expressions in resting MM T cells showed a lower ratio of CD45RA to CD45R0 in both CD4 (P less than .05) and CD8 (P less than .001) subpopulations. These data indicate that (a) some MM T cells require significantly fewer accessory signals (if any) to express the IL-2 receptor fully, secrete IL-2, and proliferate on multivalent cross-linking of the CD3/TCR complex; and (b) this peculiar state of activation is associated with high HLA-DR expression in CD8+ lymphocytes.

Aged↗
PubMed1991

Thrombin attenuates the stimulatory effect of histamine on Ca2+ entry in confluent human umbilical vein endothelial cell cultures.

Human umbilical vein endothelial cells were grown to confluence, as first passage cells, on coverslips. Treatment with ionomycin or histamine caused a sustained rise in intracellular Ca2+ (measured by Fura-2 fluorescence), but after treatment with thrombin, only a transient rise in Ca2+ was observed. Furthermore, the addition of thrombin after ionomycin or histamine lowered the raised Ca2+ back to near control levels. This effect of thrombin was concentration dependent, with increasing concentrations producing increases in both the rate and extent of the lowering of Ca2+. A similar effect of thrombin was seen on video imaging of Fura-2-loaded cell monolayers. The Ca2(+)-lowering effect of thrombin was not mimicked by phorbol 12-myristate 13-acetate nor blocked by staurosporine, indicating a lack of involvement of protein kinase C; intracellular pH also does not appear to be involved. The mechanism by which thrombin lowers cytoplasmic Ca2+ is due mainly to inhibition of Ca2+ entry since thrombin prevented the stimulated influx of Mn2+ caused by histamine or ionomycin. It may therefore be that in vivo under certain physiological or pathological conditions, thrombin's effects on intracellular Ca2+ are more transient than those of histamine, and thrombin also may induce transience in histamine's actions.

Calcium↗
PubMed1991

Three intracellular signals for cytotoxic T lymphocyte-mediated killing. Independent roles for protein kinase C, Ca2+ influx, and Ca2+ release from internal stores.

Ligation of the TCR after interaction of a CTL with a relevant target cell results in a change in CTL shape with reorientation and secretion of cytoplasmic granules. Secretion can be induced by treating CTL with the combination of a phorbol ester to activate protein kinase C and ionomycin to increase the intracellular Ca2+ concentration by ion-selective permeabilization of both the plasma membrane and internal Ca2+ storage depots. Treatment of cloned murine CTL with PMA induced a change in cell shape without movement of cytoplasmic granules, whereas increased intracellular [Ca2+] after treatment with ionomycin resulted in granule movement without shape change. Granule movement induced by ionomycin did not lead to secretion in the absence of PMA. Direct activation of the TCR resulted in increased intracellular Ca2+ predominately from influx of extracellular Ca2+ with a substantially smaller contribution from release of Ca2+ from internal stores. By independently inhibiting either component of the TCR-initiated increase in intracellular Ca2+, it was determined that both sources of Ca2+ were required for granule movement and secretion. Thus, increases in intracellular Ca2+ concentration mediated by these two sources appear not to be functionally equivalent. Taken together, these results indicate that the CTL functional response that normally occurs after interaction with an antigenically relevant target cell is regulated by three independent signals.

Animals↗
PubMed1991

Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation.

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.

Animals↗
PubMed1990

Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum.

In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.

Acetylglucosaminidase↗
PubMed1990

Muscarinic receptor-mediated translocation of calmodulin in SK-N-SH human neuroblastoma cells.

The role of muscarinic receptor-mediated polyphosphoinositide hydrolysis and subsequent calcium signals in altering the subcellular localization of calmodulin (CaM) was examined in SK-N-SH human neuroblastoma cells. Upon incubation of the cells with the full agonist carbachol, a 4.5- to 5-fold increase in CaM in the cytosol was observed, from 126 ng of CaM to 629 ng of CaM. There was an accompanying 68% decrease in membrane-bound CaM. The increase in the cytosol was maximal by 15 min, as was a corresponding decrease in membrane-associated CaM. The redistribution of CaM was maintained for at least 2 hr, before returning toward control levels by 4 hr. The EC50 values for carbachol in eliciting the translocation were 3.7 microM for the increase in cytosol and 1.3 microM for the decrease in membranes. The maximal changes in CaM concentration in both membranes and cytosol occurred with 10 microM carbachol. Incubation of the SK-N-SH cells with the partial muscarinic agonists bethanechol and arecoline resulted in 27 and 26% decreases in membrane-associated CaM, respectively, and 28 and 35% increases in cytosolic CaM, respectively. Thus, the partial agonists were less efficacious than carbachol in eliciting changes in CaM localization. Atropine completely blocked the carbachol-stimulated translocation, whereas the nicotinic agonist 1,1-dimethyl 4-phenylpiperazinium had no effect on the localization of CaM. Activation of receptors coupled to adenylate cyclase did not affect distribution of CaM. CaM content in membranes and cytosol of cells incubated with prostaglandin E1 or the alpha 2-adrenergic agonist UK 14,304 was not different from control values. The ionophore ionomycin (10 microM) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 nM) were both able to elicit changes in CaM distribution. Ionomycin caused a 64% increase in CaM in the cytosol, with no significant change in membrane CaM. TPA elicited a decrease in membrane-associated CaM, with a corresponding increase in CaM in the cytosol. When TPA and ionomycin were incubated together, the translocation was equal in magnitude to that observed with carbachol alone. The protein kinase C inhibitor H-7 blocked the TPA-stimulated response and partially blocked the carbachol-stimulated response. The CaM-binding protein neuromodulin, which demonstrates a decreased affinity for CaM in the presence of Ca2+ and when phosphorylated by protein kinase C, was present in both membranes and cytosol of SK-N-SH cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Hormone-evoked calcium release from intracellular stores is a quantal process.

Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.

Animals↗
PubMed1988

Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils.

Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗