Biomedical subjects

F B Davis

Publications and source records attributed to F B Davis.

At least 19 recordsLinked to original sources

PubMed2006

Pro-angiogenesis action of thyroid hormone and analogs in a three-dimensional in vitro microvascular endothelial sprouting model.

AIM: Our laboratory has recently demonstrated the pro-angiogenesis effects of thyroid hormone in the chick chorioallantoic membrane model. METHODS: Generation of new blood vessels from existing vessels was promoted two- to three-fold by either L-thyroxine (T4) or 3,5,3'-triiodo-L-thyronine (T3) at total hormone concentrations of T7-T9 M. RESULTS: T4-agarose, a formulation of thyroid hormone that does not cross the cell membrane, produced a potent pro-angiogenesis effect comparable to that obtained with T3 or T4. In the present investigation, T3, T4, T4-agarose, and basic fibroblast growth factor, each added to vascular endothelial growth factor, produced comparable pro-angiogenesis effects in the in vitro three-dimensional human microvascular endothelial sprouting model. The pro-angiogenesis effect of the thyroid hormone analogs was blocked by PD 98059, an inhibitor of the mitogen-activated protein kinase (MAPK; ERK1/2) signal transduction cascade. A specific avb3 integrin antagonist (XT199) also inhibited the pro-angiogenesis effect of either thyroid hormone analogs or T4-agarose. Tetrac, a thyroid hormone analog that blocks cell surface-initiated actions of T4 and T3, inhibited the pro-angiogenesis response of thyroid hormone. CONCLUSIONS: T4, T3, and T4-agarose are pro-angiogenic in the three-dimensional human microvascular endothelial sprouting model, an action that is initiated at the plasma membrane, involves avb3 integrin receptors, and is MAPK-dependent.

Cells, Cultured↗

PubMed2004

Oestrogen inhibits resveratrol-induced post-translational modification of p53 and apoptosis in breast cancer cells.

Resveratrol, a naturally occurring stilbene, induced apoptosis in human breast cancer MCF-7 cells. The mechanism of this effect was dependent on mitogen-activated protein kinase (MAPK, ERK1/2) activation and was associated with serine phosphorylation and acetylation of p53. Treatment of MCF-7 cells with resveratrol in the presence of 17beta-oestradiol (E(2)) further enhanced MAPK activation, but E(2) blocked resveratrol-induced apoptosis, as measured by nucleosome ELISA and DNA fragmentation assays. E(2) inhibited resveratrol-stimulated phosphorylation of serines 15, 20 and 392 of p53 and acetylation of p53 in a concentration- and time-dependent manner. These effects of E(2) on resveratrol action were blocked by ICI 182,780 (ICI), an inhibitor of the nuclear oestrogen receptor-alpha (ER). ICI 182,780 did not block the actions of resveratrol, alone. Electrophoretic mobility studies of p53 binding to DNA and of p21 expression indicated that E(2) inhibited resveratrol-induced, p53-directed transcriptional activity. These results suggest that E(2) inhibits p53-dependent apoptosis in MCF-7 cells by interfering with post-translational modifications of p53 which are essential for p53-dependent DNA binding and consequent stimulation of apoptotic pathways. These studies provide insight into the complex pathways by which apoptosis is induced by resveratrol in E(2)-depleted and -repleted environments.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Comparison of the mechanisms of nongenomic actions of thyroid hormone and steroid hormones.

Steroids and thyroid hormone are thought primarily to act via binding to hormone-specific nuclear receptor superfamily members. The nuclear ligand-receptor complexes then initiate transcriptional activity. Actions of steroids and iodothyronines that are nongenomic or extranuclear in mechanism have been recognized recently and new insights into such mechanisms are available. Despite their distinct structures and biologic effects, the two families of hormones have similarities in the mechanisms of their nongenomic actions. That is, both steroids and thyroid hormone appear to interact with specific cell surface G protein-coupled receptors and to activate signal transducing kinases such as those involved in the mitogen-activated protein kinase (MAPK) pathway. Much is known about the ability of certain steroids such as estrogen and mineralocorticoids to increase [Ca2+]i acutely and stimulation of the MAPK cascade by L-T4 appears to depend upon a hormone-induced increase in [Ca2+]i via phosphoinositide pathway activation. At least in the case of iodothyronines, hormone activation of the MAPK pathway modulates the cellular activities of certain cytokines and growth factors. One of the two cell surface estrogen receptors (ERs) may be an expression of the same transcript as that for nuclear ER, whereas the mineralocorticoid and progesterone-binding proteins in the plasma membrane appear to be products of genes different from those of nuclear receptors. Iodothyronine structure-activity relationships at the plasma membrane binding site for thyroid hormone suggest that the cell surface receptor for T4 that also binds 3,5,3'-triiodo-L-T3 is different from the nuclear T3 receptor (TR). There are interfaces of nongenomic and genomic mechanisms for both steroids and thyroid hormone. For example, by nongenomic mechanisms, estrogen and thyroid hormone can promote serine phosphorylation, respectively, of nuclear ER and TR. Transcriptional activity of the nuclear receptor proteins can be altered by such phosphorylation.

Animals↗

PubMed2001

Thyroid hormone promotes serine phosphorylation of p53 by mitogen-activated protein kinase.

L-Thyroxine (T(4)) nongenomically promotes association of mitogen-activated protein kinase (MAPK) and thyroid hormone receptor TRbeta1 (TR) in the cell nucleus, leading to serine phosphorylation of the receptor. The oncogene suppressor protein, p53, is serine phosphorylated by several kinases and is known to interact with TRbeta1. We studied whether association of p53 and TR is modulated by T(4) and involves serine phosphorylation of p53 by MAPK. TR-replete 293T human kidney cells were incubated with a physiological concentration of T(4) for 10-90 min. Nuclear fractions were immunoprecipitated and the resulting proteins separated and immunoblotted for co-immunoprecipitated proteins. Activated MAPK immunoprecipitates of nuclei from T(4)-treated cells accumulated p53 in a time-dependent manner; T(4) and T(4)-agarose were more effective than T(3). T(4)-induced nuclear complexing of p53 and MAPK was inhibited by PD 98059 (PD) and U0126, two MAPK kinase (MEK) inhibitors, and was absent in cells treated with MEK antisense oligonucleotide and in dominant negative Ras cells. T(4) also caused nuclear co-immunoprecipitation of TRbeta1 and p53, an effect also inhibited by PD. Nuclear complexing of p53 and MAPK also occurred in HeLa cells, which lack functional TR. Constitutively activated MAPK caused phosphorylation of a recombinant p53-GST fusion protein in vitro; thus, p53 is a substrate for MAPK. An indicator of p53 transcriptional activity, accumulation of the immediate-early gene product, c-Jun, was inhibited by T(4). This T(4) effect was reversed by PD, indicating that the transcriptional activity of p53 was altered by T(4)-directed MAPK-p53 interaction.

3T3 Cells↗

PubMed2000

Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR.

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.

Cell Line↗

PubMed2000

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB.

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.

Benzoquinones↗

PubMed1999

Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells.

We have examined the effects of l-thyroxine (T4) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal growth factor (EGF). T4, at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10-20 min. Activation by T4 of STAT3 was maximal at 30 min (15+/-4-fold enhancement; mean+/-S.E.M.) in 18 experiments. This effect was reproduced by T4-agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T4. In the nuclear fraction of T4-treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin fibroblasts (BG-9). T4 also potentiated the EGF-induced nuclear translocation of activated STAT1alpha and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T4 of STAT3, and the potentiation of EGF by T4, require activities of PKC, PTK and an intact MAPK pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells.

Thyroid hormone [L-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5'-O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4 and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1alpha by T4; they also showed reduced T4 potentiation of the antiviral action of interferon-gamma (IFN-gamma). T4 treatment caused tyrosine-phosphorylated MAPK-STAT1alpha nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1alpha, in the presence or absence of IFN-gamma. STAT1alpha-deficient cells transfected with STAT1alpha containing an alanine-for-serine substitution at residue 727 (STAT1alphaA727) showed minimal T4-stimulated STAT1alpha activation. IFN-gamma induced the antiviral state in cells containing wild-type STAT1alpha (STAT1alphawt) or STAT1alphaA727; T4 potentiated IFN-gamma action in STAT1alphawt cells but not in STAT1alphaA727 cells. T4-directed STAT1alpha Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1alpha activation and enhanced IFN-gamma activity.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Potentiation by thyroid hormone of human IFN-gamma-induced HLA-DR expression.

We have investigated the mechanism by which thyroid hormone potentiates IFN-gamma-induced HLA-DR expression. IFN-gamma-induced HLA-DR expression requires activation of STAT1alpha and induction of the Class II trans-activator, CIITA. HeLa and CV-1 cells treated only with L-thyroxine (T4) demonstrated increased tyrosine phosphorylation and nuclear translocation (= activation) of STAT1alpha; this hormone effect on signal transduction, and T4 potentiation of IFN-gamma-induced HLA-DR expression, were blocked by the inhibitors CGP 41251 (PKC) and genistein (tyrosine kinase). Treatment of cells with T4-agarose also caused activation of STAT1alpha. In the presence of IFN-gamma, T4 enhanced cytokine-induced STAT1alpha activation. Potentiation by T4 of IFN-gamma action was associated with increased mRNA for both CIITA and HLA-DR, with peak enhancement at 16 h (CIITA), and 2 d (HLA-DR). T4 increased IFN-gamma-induced HLA-DR protein 2.2-fold and HLA-DR mRNA fourfold after 2 d. Treatment with actinomycin D after induction of HLA-DR mRNA with IFN-gamma, with or without T4, showed that thyroid hormone decreased the t(1/2) of mRNA from 2.4 to 1.1 h. HeLa and CV-1 cells lack functional nuclear thyroid hormone receptor. Tetraiodothyroacetic acid (tetrac) and 3,5,3'-triiodo-thyroacetic acid (triac) blocked T4 potentiation of IFN-gamma-induced HLA-DR expression and T4 activation of STAT1alpha. These studies define an early hormone recognition step at the cell surface that is novel, distinct from nuclear thyroid hormone receptor, and blocked by tetrac and triac. Thus, thyroid hormone potentiation of IFN-gamma-induced HLA-DR transcription is mediated by a cell membrane hormone binding site, enhanced activation of STAT1alpha, and increased CIITA induction.

Biological Transport↗

PubMed1998

Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin.

Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase.

Chemokine CCL4↗

PubMed1997

Protein synthesis-dependent potentiation by thyroxine of antiviral activity of interferon-gamma.

We have studied the prenuclear signal transduction pathway by which thyroid hormone potentiates the antiviral activity of human interferon-gamma (IFN-gamma) in HeLa cells, which are deficient in thyroid hormone receptor (TR). The action of thyroid hormone was compared with that of milrinone, which has structural homologies with thyroid hormone. L-Thyroxine (T4), 3,5,3'-L-triiodothyronine (T3), and milrinone enhanced the antiviral activity of IFN-gamma up to 100-fold, a potentiation blocked by cycloheximide. The 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil did not block the T4 effect. 3,3',5,5'-Tetraiodothyroacetic acid prevented the effect of T4 but not of milrinone. The effects of T4 and milrinone were blocked by inhibitors of protein kinases C (PKC) and A (PKA) and restored by PKC and PKA agonists; only the effect of T4 was blocked by genistein, a tyrosine kinase inhibitor. In separate models, milrinone was shown not to interact with nuclear TR-beta. T4 potentiation of the antiviral activity of IFN-gamma requires PKC, PKA, and tyrosine kinase activities but not traditional TR.

Amrinone↗

PubMed1996

Thyroid hormone analogues potentiate the antiviral action of interferon-gamma by two mechanisms.

L-thyroxine (L-T4) potentiates the antiviral activity of human interferon-gamma (IFN-gamma) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN-gamma (1.0 IU/ml), 2) for 24 h cotreatment with IFN-gamma, 3) for 4, after 20 h cell incubation with IFN-gamma, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN-gamma. The antiviral effect of IFN-gamma was then assayed. L-T4 potentiated the antiviral action of IFN-gamma by a reduction in virus yield of more than two logs, the equivalent of a more than 100-fold potentiation of the IFN's antiviral effect. 3,3 of the IFN's antiviral effect. 3,3',5-L-triiodothyronine (L-T3) was as effective as L-T4 when coincubated for 24 h with IFN-gamma but was less effective than L-T4 when coincubated for only 4 h. D-T4, D-T3, 3,3',5-triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5-diiodothyronine (T2) were inactive. When preincubated with L-T4 for 24 h prior to IFN-gamma treatment, tetrac blocked L-T4 potentiation, but, when coincubated with L-T4 for 4 h after 20 h IFN-gamma, tetrac did not inhibit the L-T4 effect. 3,3',5-L-triiodothyronine (rT3) also potentiated the antiviral action of IFN-gamma, but only in the preincubation model. Furthermore, the effects of rT3 preincubation and L-T3 coincubation were additive, resulting in 100-fold potentiation of the IFN-gamma effect. When L-T4, L-T3, or rT3, plus cycloheximide (5 micrograms/ml), was added to cells for 24 h and then removed prior to 24 h IFN-gamma exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN-gamma potentiation by 4 h of L-T4 or L-T3 coincubation was not inhibited by cycloheximide (25 micrograms/ml). These studies demonstrate two mechanisms by which thyroid hormones can potentiate IFN-gamma's effect: 1) a protein synthesis-dependent mechanism evidenced by enhancement of IFN-gamma's antiviral action by L-T4, L-T3, or rT3 preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis-independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L-T4 or L-T3, but not rT3, with IFN-gamma. The protein synthesis-dependent pathway is responsive to rT3, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN-gamma can be regulated has not previously been described.

Antiviral Agents↗

PubMed1996

Potentiation by thyroxine of interferon-gamma-induced HLA-DR expression is protein kinase A- and C-dependent.

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) potentiate the antiviral state induced by interferon-gamma(IFN-gamma) in homologous cells by a mechanism that is dependent upon calcium/phospholipid-dependent protein kinase (PKC). L-T4 and T3 also potentiate induction by IFN-gamma of MHC class II HLA-DR antigen expression in HeLa cells. In the present studies of HLA-DR expression, the PKC inhibitor staurosporine (0.1-1 nM) enhanced the expression of HLA-DR when the inhibitor was added simultaneously with IFN-gamma, 100 IU/ml. In the presence of IFN-gamma and 10(-7) M T4, the same concentrations of staurosporine inhibited potentiation of HLA-DR expression by thyroid hormone. A more specific PKC inhibitor, CGP41251 (0.5-5 nM), similarly enhanced HLA-DR expression in the presence of IFN-gamma but inhibited thyroid hormone potentiation of antigen expression. Both actions of CGP41251 were suppressed when cells were also treated with phorbol 12-myristate 13-acetate (PMA). A phospholipase C inhibitor, U73122 (1-1000 nM), did not alter the potentiating ability of T4, although it inhibited in a concentration-dependent manner the expression of HLA-DR induced by IFN-gamma. The potentiating effect of T4 was much more sensitive to a cyclic AMP-dependent protein kinase (PKA) inhibitor,KT5720 (1-1000nM), than was the induction of HLA-DR by IFN-gamma. The inhibitory effects of KT5720 were reversed by concurrent 8-bromo-cAMP treatment. The calmodulin antagonist W-7 (5-50 microM) did not alter IFN-gamma induction of HLA-DR in either the presence or absence of T4. HLA-DR expression in HeLa cells appears to be under PKC-associated inhibition; IFN-gamma reverses this inhibition to promote the appearance of the DR antigen. In contrast, potentiation by T4 of induction of HLA-DR by IFN-gamma requires activation of PKC. PKA is involved both in DR induction by IFN-gamma and in potentiation of the latter by T4. Thus, PKA and PKC have discrete roles in IFN-gamma-induced MHC class II antigen expression and its modulation by thyroid hormone.

Antiviral Agents↗

PubMed1996

Nongenomic actions of thyroid hormone.

Nongenomic actions of thyroid hormone are by definition independent of nuclear receptors for the hormone and have been described at the plasma membrane, various cell organelles, the cytoskeleton, and in cytoplasm. The actions include alterations in solute transport (Ca2+, Na+, glucose), changes in activities of several kinases, including protein kinase C, cAMP-dependent protein kinase and pyruvate kinase M2 (PKM2), effects on efficiency of specific mRNA translation and mRNA t1/2, modulation of mitochondrial respiration, and regulation of actin polymerization (promotion of formation of F-actin). Iodothyronines also can regulate nongenomically the state of contractile elements in vascular smooth muscle cells (VSMC). The physiologic significance at the cellular level of certain of these actions has been demonstrated, for example, in the cases of myocardiocyte Na+ current, red cell Ca2+ content, and the control by hormone-induced alterations in actin solubility of cell surface activity of iodothyronine 5'-monodeiodinase activity and the intracellular distribution of protein disulfide isomerase activity. The physiologic significance of these actions at the organ or system level is less clear, but extranuclear effects of thyroid hormone on myocardial Na+ channel, sarcoplasmic reticulum Ca(2+)-ATPase activity, and contractile state of VSMC may each contribute to acute effects of thyroid hormone on cardiac output that have recently been described clinically. The molecular mechanisms for nongenomic actions are incompletely understood; relevant binding sites and signal transduction pathways have been described for hormone actions on plasma membrane Ca(2+)-ATPase activity, and PKM2 monomer is known to bind T3 and, as a result, prevent activation of the kinase via tetramer formation. Nongenomic actions of thyroid hormone may have different structure-activity relationships of iodothyronines from those effects that depend upon nuclear receptors; they may have different time courses and may invoke complex signal transduction pathways before the action is detected.

Animals↗

PubMed1996

Potentiation by thyroxine of interferon-gamma-induced antiviral state requires PKA and PKC activities.

Added to HeLa cells previously exposed to recombinant human interferon (IFN)-gamma for 20 h, thyroid hormone [L-thyroxine (T4)] in physiological concentrations potentiates the antiviral action of IFN-gamma by more than 100-fold in 4 h. We examined protein kinase activities for their contributions to the mechanism of this posttranslational effect of thyroid hormone. Added concurrently with thyroid hormone, the protein kinase C (PKC) inhibitor CGP-41251 (5 nM) blocked T4 potentiation of IFN-gamma action. Coincubated with CGP-41251, phorbol 12-myristate 13-acetate (PMA) reversed the effect of the inhibitor on thyroid hormone action. U-73122 (10 nM), a phospholipase C inhibitor, also blocked hormone potentiation. KT-5720 (500 nM), a protein kinase A (PKA) inhibitor, completely inhibited the T4 effect, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) restored hormone action in the presence of KT-5720. In the absence of T4, 8-BrcAMP and PMA, added together to cells in the 4-h paradigm, fully reproduced hormone potentiation of the antiviral effect of IFN-gamma. Incubated individually with IFN-gamma-treated cells, the two agonists had no potentiating action. Thyroid hormone apparently must activate both PKA and PKC in the nongenomic pathway of IFN-gamma action to enhance antiviral activity in HeLa cells.

Animals↗

PubMed1995

Structure-activity relationships of milrinone analogues determined in vitro in a rabbit heart membrane Ca(2+)-ATPase model.

The cardiac activity of a series of analogues of the positive inotropic bipyridines amrinone (5-amino-[3,4'-bipyridin]-6(1H)-one) and milrinone (2-methyl-5-cyano-[3,4'-bipyridin]-6(1H)-one) was evaluated in vitro in a rabbit myocardial membrane Mg(2+)-dependent, Ca(2+)-stimulable adenosine triphosphatase (Ca(2+)-ATPase) model and structure-activity relationships were compared for nine closely related derivatives. In the present studies, a 5-bromo analogue of milrinone stimulated myocardial membrane Ca(2+)-ATPase significantly (10(-7) M; P < 0.001 vs control, with 67% of the activity of milrinone), whereas a 2'-methyl-2H-milrinone derivative was inactive. Although amrinone was inactive in this assay, its 2-methyl analogue was stimulatory. However, analogues lacking a 2-substituent (with or without a 5-cyano group) or with the 3-N position blocked by a methyl group did not stimulate myocardial membrane Ca(2+)-ATPase activity. Structural data for these bipyridines show that those with either a 2- or 2'-methyl substituent have a twist conformation, whereas those without are nearly planar. Activity data reveal that those bipyridines with a nonplanar conformation are more active in the Ca(2+)-ATPase assay. Further study of milrinone analogues with a 2'-methyl substituent shows that even though the effect on the twist angle is equivalent to that of 2-methyl substitution, these analogues are less potent. Data for this series reveal that the prerequisites for Ca(2+)-ATPase stimulation include not only a 2-methyl to maintain a twist conformation but also a free 3-N position and a 5-substituent. This model for optimal activity in the myocardial membrane Ca(2+)-ATPase system differs from those proposed for phosphodiesterase enzyme receptor recognition only in the requirement for a nonplanar molecule. We have previously shown that milrinone, but not amrinone, shares structural homology with thyroxine and was able to stimulate myocardial membrane Ca(2+)-ATPase activity in a manner similar to the thyroid hormone. Additionally, milrinone, but not amrinone, was an effective competitor for thyroxine binding to the serum transport protein transthyretin. Analysis of the milrinone-transthyretin crystal complex confirms the structural homology between milrinone and thyroid hormone which is not shared by amrinone. Modeling studies of the binding interactions of milrinone analogues indicate that the 2-desmethylmilrinone analogue, the most inhibitory analogue, lacks the hydrophobic contacts present in milrinone in its transthyretin-bound complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Amrinone↗

PubMed1995

Inositol phosphates modulate human red blood cell Ca(2+)-adenosine triphosphatase activity in vitro by a guanine nucleotide regulatory protein.

D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] inhibits human red blood cell (RBC) Ca(2+)-stimulable, Mg(2+)-dependent adenosine triphosphatase (Ca(2+)-ATPase) activity in vitro. Because we have previously shown that adrenergic receptors exist on the human mature RBC membrane and can modulate Ca(2+)-ATPase activity, we examined the possibility that a guanine nucleotide regulatory protein (G protein) mediated the Ins(1,4,5)P3 effect. Guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) 10(-4) mol/L also inhibited RBC Ca(2+)-ATPase activity. Pertussis toxin 200 ng/mL blocked the effects of both Ins(1,4,5)P3 and GTP gamma S on Ca(2+)-ATPase activity. In separate studies, pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation was shown to occur in RBC membranes under conditions in which measurements of Ca(2+)-ATPase activity were performed. When Ins(1,4,5)P3 10(-7) mol/L and GTP gamma S 10(-6) mol/L were added to membranes concurrently, their inhibitory actions on the enzyme were additive. At greater concentrations of Ins(1,4,5)P3 (10(-6) to 10(-5) mol/L) and GTP gamma S (10(-4) mol/L), the inositol phosphate reversed the inhibitory effect of GTP gamma S. These observations indicate that the novel effect of Ins(1,4,5)P3 on the activity of a plasma membrane Ca(2+)-ATPase depends at least in part on the action of a pertussis toxin-susceptible G protein.

Adult↗

PubMed1994

Retinoic acid inhibition of thyroxine binding to human transthyretin.

All-trans retinoic acid is a potent inhibitor of [125I]-thyroxine (T4) binding to human erythrocyte membranes and can block the activation by thyroid hormone of erythrocyte Ca(2+)-ATPase [J. Biol. Chem. (1989) 264, 687-689]. In the present studies, retinoic acid was examined for its ability to displace thyroxine from binding sites on human transthyretin (TTR). Scatchard analysis of [125I]T4 binding to purified TTR, determined by equilibrium dialysis, revealed two classes of binding sites with association constants of 3.2 x 10(9) M-1 and 8.1 x 10(6) M-1. All-trans retinoic acid also displaced [125I]T4; 40% of the specifically bound [125I]T4 was displaced at a retinoic acid concentration of 2 x 10(-5) M. Analysis of the high affinity T4 binding site suggests that the Ka for retinoic acid to that site is approx. 10(7) M-1. 8-Anilinonaphthalene-1-sulfonate (ANS), a strongly fluorescing dye, binds to the thyroxine binding sites on TTR. T4 and 3,5,3'-L-triiodothyronine (T3) shifted the fluorescence emission maximum and intensity of an ANS-TTR solution toward the spectrum obtained from uncomplexed ANS. All-trans retinoic acid caused a similar shift in the emission spectrum of ANS, but was less potent than T4. Retinol failed to quench the emission intensity of the ANS-TTR complex, while 13-cis-retinoic acid was less effective than all-trans retinoic acid.

Anilino Naphthalenesulfonates↗