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Biomedical subjects

M Schwaninger

Publications and source records attributed to M Schwaninger.

At least 37 records · Page 2Linked to original sources
PubMed2000

Adenosine-induced expression of interleukin-6 in astrocytes through protein kinase A and NF-IL-6.

In various neurologic diseases, astrocytes express interleukin-6 (IL-6), which is an endogenous pyrogen, a neuroprotective factor, and a regulator of the blood-brain barrier. The expression of IL-6 in astrocytes is stimulated by extracellular adenosine through A(2B) receptors. To investigate the signaling cascade that induces IL-6 gene transcription further, we transfected primary mouse astrocytes with a reporter gene construct, in which luciferase expression is directed by the human IL-6 promoter. Expression of PKI, an inhibitor of protein kinase A (PKA), interfered with IL-6 transcription indicating that PKA mediates the effect of adenosine. The CAAT box of the IL-6 promoter is necessary for the stimulation by adenosine as a mutation in this element reduced the stimulation by adenosine. Indeed, the cAMP agonist forskolin increased the binding of the transcription factors NF-IL-6 and C/EBPdelta to the CAAT box of the IL-6 promoter in nuclear extracts of astrocytes. Inhibition of the de novo synthesis of NF-IL-6 by cycloheximide or an antisense oligonucleotide reduced the enhancement of NF-IL-6 binding to the CAAT box and inhibited stimulation of IL-6 transcription by forskolin. In addition, overexpression of NF-IL-6 induced IL-6 transcription. This suggests that adenosine induces the de novo synthesis of NF-IL-6 through activation of PKA and thereby stimulates transcription of IL-6 in astrocytes.

Adenosine↗
PubMed2000

Elevated plasma concentrations of lipoprotein(a) in medicated epileptic patients.

Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for vascular diseases. There are no data on Lp(a) levels in patients on long-term medication with carbamazepine, phenytoin, phenobarbital, or valproate. To investigate the effects of such treatment on Lp(a) levels and common carotid artery intima media thickness we studied 51 epileptic outpatients on long-term antiepileptic medication and 51 age-and sex-matched controls. Lp(a) levels above 45 mg/dl were found in 11 of 50 patients, but in only 4 of 51 controls (P < 0.05). The mean serum concentration of Lp(a) was 33.0+/-7.0 mg/dl in patients and 16.9+/-2.7 mg/dl in controls (P < 0.05). Epileptic patients also had a thicker intima media of the common carotid artery (0.79+/-0.04 mm) than controls (0.69+/-0.02 mm, P < 0.05) as measured by B-mode ultrasonography. Our results suggest an untoward effect of long-term antiepileptic medication on Lp(a) serum concentrations. Elevated Lp(a) levels might be a risk factor for arteriosclerosis in epileptic patients.

Adult↗
PubMed2000

Intravenous brain-derived neurotrophic factor reduces infarct size and counterregulates Bax and Bcl-2 expression after temporary focal cerebral ischemia.

BACKGROUND AND PURPOSE: Pretreatment with intraventricular brain-derived neurotrophic factor (BDNF) reduces ischemic damage after focal cerebral ischemia. In this experiment we studied the effect of intravenous BDNF delivered after focal cerebral ischemia on neurological outcome, infarct size, and expression of proapoptotic and antiapoptotic proteins Bax and Bcl-2, respectively. METHODS: With the use of the suture occlusion technique, the right middle cerebral artery in rats was temporarily occluded for 2 hours. Thirty minutes after vessel occlusion, BDNF (300 microg/kg per hour in vehicle; n=12) or vehicle alone (n=13) was continuously infused intravenously for 3 hours. After 24 hours the animals were weighed and neurologically assessed on a 5-point scale. The animals were then killed, and brains underwent either 2,3,5-triphenyltetrazolium chloride staining for assessment of infarct volume or paraffin embedding for morphology and immunohistochemistry (Bax, Bcl-2). RESULTS: Physiological parameters (mean arterial blood pressure, PO(2), PCO(2), pH, body temperature, glucose) and weight revealed no difference between groups. Neurological deficit was improved in BDNF-treated animals versus controls (P:<0.05, unpaired, 2-tailed t test). Mean+/-SD infarct volume was 229.7+/-97.7 mm(3) in controls and 121.3+/-80.2 mm(3) in BDNF-treated animals (P:<0.05, unpaired, 2-tailed t test). Cortical infarct volume was 155.5+/-78.5 mm(3) in the placebo group and 69.9+/-50.2 mm(3) in the BDNF-treated group (P:<0.05, unpaired, 2-tailed t test). Subcortical infarct volume was 74.1+/-30.6 mm(3) in the placebo group and 51.1+/-26.8 mm(3) in the BDNF-treated group (P:=NS). Bax-positive neurons were significantly reduced in the ischemic penumbra in BDNF-treated animals (P:<0.05, unpaired, 2-tailed t test), whereas Bcl-2-positive neurons were significantly increased in this area (P:<0.001, unpaired, 2-tailed t test). CONCLUSIONS: This study demonstrates a neuroprotective effect of BDNF when delivered intravenously after onset of focal cerebral ischemia. As shown here, one possible mechanism of action of neuroprotection of BDNF after focal ischemia appears to be counterregulation of Bax/Bcl-2 proteins within the ischemic penumbra.

Animals↗
PubMed1999

Characterization of a novel calcium response element in the glucagon gene.

To maintain blood glucose levels within narrow limits, the synthesis and secretion of pancreatic islet hormones is controlled by a variety of extracellular signals. Depolarization-induced calcium influx into islet cells has been shown to stimulate glucagon gene transcription through the transcription factor cAMP response element-binding protein that binds to the glucagon cAMP response element. By transient transfection of glucagon-reporter fusion genes into islet cell lines, this study identified a second calcium response element in the glucagon gene (G2 element, from -165 to -200). Membrane depolarization was found to induce the binding of a nuclear complex with NFATp-like immunoreactivity to the G2 element. Consistent with nuclear translocation, a comigrating complex was found in cytosolic extracts of unstimulated cells, and the induction of nuclear protein binding was blocked by inhibition of calcineurin phosphatase activity by FK506. A mutational analysis of G2 function and nuclear protein binding as well as the effect of FK506 indicate that calcium responsiveness is conferred to the G2 element by NFATp functionally interacting with HNF-3beta binding to a closely associated site. Transcription factors of the NFAT family are known to cooperate with AP-1 proteins in T cells for calcium-dependent activation of cytokine genes. This study shows a novel pairing of NFATp with the cell lineage-specific transcription factor HNF-3beta in islet cells to form a novel calcium response element in the glucagon gene.

Base Sequence↗
PubMed1999

NF-kappaB is activated and promotes cell death in focal cerebral ischemia.

The transcription factor NF-kappaB is a regulator of cell death or survival. To investigate the role of NF-kappaB in neuronal cell death, we studied its activation in a rodent model of stroke. In the ischemic hemisphere, NF-kappaB was activated, as determined by increased expression of an NF-kappaB-driven reporter transgene, nuclear translocation of NF-kappaB in neurons and enhanced DNA binding of NF-kappaB subunits RelA and p50. In p50 knockout mice, ischemic damage was significantly reduced. This indicates a cell death-promoting role of NF-kappaB in focal ischemia. NF-kappaB may provide a new pharmacological target in neurologic disease.

Animals↗
PubMed1999

Bradykinin induces interleukin-6 expression in astrocytes through activation of nuclear factor-kappaB.

Bradykinin, a mediator of inflammation, is produced in the brain during trauma and stroke. It is thought to open the blood-brain barrier, although the mechanism is unclear. We have investigated, therefore, the effect of bradykinin on the expression of interleukin-6 (IL-6), a putative modulator of the blood-brain barrier, in astrocytes. IL-6 gene transcription was evaluated by transient transfection of the human IL-6 promoter linked to the luciferase gene. In murine astrocytes, bradykinin stimulated IL-6 secretion and gene transcription. The effect of bradykinin was blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases, and by bisindolylmaleimide I, an inhibitor of protein kinase C, suggesting the involvement of these protein kinases. Mutations in the multiple response element and the binding site for nuclear factor-kappaB (NF-kappaB), but not in other known elements of the IL-6 promoter, interfered with induction of IL-6 transcription. The involvement of NF-kappaB was supported further by the finding that overexpression of nmIkappaB alpha, a stable inhibitor of NF-kappaB, inhibited the induction of IL-6 by bradykinin. Bradykinin activated NF-kappaB in primary astrocytes as shown by increased DNA binding of NF-kappaB. These data demonstrate that bradykinin stimulates IL-6 expression through activation of NF-kappaB, which may explain several inflammatory effects of bradykinin.

Animals↗
PubMed1999

Elevated plasma concentrations of homocysteine in antiepileptic drug treatment.

PURPOSE: Homocysteine is an experimental convulsant and an established risk factor in atherosclerosis. A nutritional deficiency of vitamin B6, vitamin B12, or folate leads to increased homocysteine plasma concentrations. During treatment with carbamazepine (CBZ), phenytoin, or phenobarbital, a deficiency in these vitamins is common. The objective of the study was to test the hypothesis that antiepileptic drug (AED) treatment is associated with increased homocysteine plasma concentrations. METHODS: A total of 51 consecutive outpatients of our epilepsy clinic receiving stable, individually adjusted AED treatment and 51 sex- and age-matched controls were enrolled in the study. Concentrations of total homocysteine and vitamin B6 were measured in plasma; vitamin B12 and folate were measured in the serum of fasted subjects. RESULTS: Patients and controls differed significantly in concentrations of folate ( 13.5+/-1.0 vs. 17.4+/-0.8 nM and vitamin B6 (39.7+/-3.4 vs. 66.2+/-7.5 nM), whereas serum concentrations of vitamin B12 were similar. The homocysteine plasma concentration was significantly increased to 14.7+/-3.0 microM in patients compared with controls (9.5+/-0.5 microM; p < 0.05, Wilcoxon rank-sum test). The number of patients with concentrations of >15 microM was significantly higher in the patient group than among controls. The same result was obtained if only patients with CBZ monotherapy were included. Patients with increased homocysteine plasma concentrations had lower folate concentrations. CONCLUSIONS: These data support the hypothesis that prolonged AED treatment may increase plasma concentrations of homocysteine, although the alternative explanation that increased homocysteine plasma concentrations are associated with the disease and not the treatment cannot be completely excluded at the moment.

Age Factors↗
PubMed1999

Inhibition of cyclic AMP response element-binding protein/cyclic AMP response element-mediated transcription by the immunosuppressive drugs cyclosporin A and FK506 depends on the promoter context.

The immunosuppressants cyclosporin A and FK506 (tacrolimus) can block the phosphatase calcineurin, thereby inhibiting gene transcription directed by the cyclic AMP (cAMP)- and calcium-responsive transcription factor, cAMP response element (CRE)-binding protein, and its binding site, CRE, in various cell lines. This action is a novel molecular mechanism of cyclosporin A and FK506 action. Because inhibition of CREB/CRE-directed transcription by cyclosporin A and FK506 has previously been observed by using synthetic minienhancers, reporter fusion genes were constructed to examine the effect of cyclosporin A and FK506 on the transcriptional activity of CRE-containing natural promoters. In transient transfection experiments, cyclosporin A and FK506 inhibited the transcriptional activation by cAMP and the membrane depolarization of three CRE-containing promoters. However, cyclosporin A and FK506 failed to inhibit the activation by cAMP of another promoter, the rat insulin I gene promoter. The lack of cyclosporin A/FK506 sensitivity is not intrinsic to the insulin CRE because cyclosporin A and FK506 inhibited the activation by cAMP of the insulin CRE when isolated and used as a synthetic minienhancer. Rather, cyclosporin A/FK506 resistance may be conferred by specific promoter interactions because a mutational analysis of the insulin promoter revealed that inside this promoter, CRE activity depends on an adjacent control element. These data show that cyclosporin A and FK506 can inhibit CRE activity when the CRE resides in its natural promoter. However, the cyclosporin A/FK506 sensitivity depends on the specific promoter context. The results suggest that cyclosporin A and FK506 may alter target tissue function through the regulation of a subset of CRE-containing genes.

Animals↗
PubMed1997

Inhibition of CREB- and cAMP response element-mediated gene transcription by the immunosuppressive drugs cyclosporin A and FK506 in T cells.

The clinically important immunosuppressant drugs cyclosporin A and FK506 (tacrolimus) inhibit in T-cells calcineurin phosphatase activity and nuclear translocation of the cytosolic component of the transcription factor nuclear factor of activated T-cells (NF-ATc) that is involved in the induction of early genes during T-cell activation. This effect has been proposed to explain at least part of the immunosuppressive effect of these drugs. Previous studies in pancreatic islet cell lines have shown that cyclosporin A and FK506 through inhibition of calcineurin interfere also with the function of the transcription factor cAMP response element binding protein (CREB) that is activated by cAMP and calcium signals and binds to cAMP/calcium response elements (CRE). By transient expression of CRE-reporter genes or GAL4-CREB fusion proteins, the present study shows that inhibition of CREB/CRE-directed transcription by cyclosporin A and FK506 occurs in a great variety of cell types including in cell lines derived from tissues in which adverse effects of the immunosuppressants develop. CREB activity and CRE-mediated transcription was blocked by these drugs also in Jurkat T-cells. When taken together with recent evidence for an essential role of CREB in T-cell activation and proliferation, the present results suggest that inhibition of CREB/CRE-directed transcription may be a molecular mechanism of the immunosuppressive effect of cyclosporin A and FK506.

Animals↗
PubMed1997

Stimulation of interleukin-6 secretion and gene transcription in primary astrocytes by adenosine.

During cerebral ischemia, the expression of interleukin-6 (IL-6), which has neuroprotective properties, increases. To understand the underlying mechanism, the regulation of IL-6 expression by neurotransmitters that accumulate during cerebral ischemia was investigated. Adenosine stimulated IL-6 secretion in primary astrocytes four- to 10-fold. The effect was concentration dependent, the EC50 being approximately 8 microM. Although the nonselective analogue 2-chloroadenosine (2CA) increased IL-6 secretion to a similar extent, the A1-selective agonist N6-cyclopentyladenosine or the A2a agonist CGS-21680 had only a marginal effect on IL-6 secretion. IL-6 secretion stimulated by 2CA (10 microM) was inhibited by the nonselective adenosine antagonist 8-(p-sulfophenyl)theophylline, whereas the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine or the A2a-selective antagonist 8-(3-chlorostyryl)caffeine had no effect, to a concentration of 0.1 microM. Transcription of the IL-6 gene was investigated by transfecting primary astrocytes with a reporter fusion gene containing the human IL-6 promoter (-179/+12). 2CA stimulated IL-6 gene transcription 2.5-fold. Mutations of the binding site for NF-kappaB or NF-IL6 abrogated the response to 2CA. Thus, an increase of extracellular adenosine during focal cerebral ischemia may stimulate IL-6 expression via A2b receptors. The induction of IL-6 expression appears to involve a transcriptional effect that depends on NF-kappaB and NF-IL6.

2-Chloroadenosine↗
PubMed1997

Characterization of a novel protein kinase C response element in the glucagon gene.

To maintain glucose levels in blood within narrow limits, the synthesis and secretion of pancreatic islet hormones are controlled by a variety of neural, hormonal, and metabolic messengers that act through multiple signal transduction pathways. Glucagon gene transcription is stimulated by cyclic AMP and depolarization-induced calcium influx. In this study, the effect of protein kinase C on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into the glucagon-producing islet cell line alphaTC2, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated glucagon gene transcription. By 5' deletions, 3' deletions, internal deletion, and oligonucleotide cassette insertion, the TPA-responsive element was mapped to the G2 element (from -165 to -200). Like TPA, overexpression of oncogenic Ras (V-12 Ras) stimulated G2-mediated transcription whereas overexpression of a dominant negative Ras mutant (N-17 Ras) blocked the effect of TPA. A mutational analysis of G2 function and nuclear protein binding indicated that protein kinase C and Ras responsiveness is conferred to the glucagon gene by HNF-3beta functionally interacting with a protein that binds to a closely associated site with sequence similarity to binding sites of Ets family proteins. HNF-3beta belongs to the winged-helix family of transcription factors and has been implicated in the control of cell-specific and developmental gene expression. The results of the present study show that the cell lineage-specific transcription factor HNF-3beta is an essential component of a novel protein kinase C response element in the glucagon gene.

Animals↗
PubMed1996

Calcium-mobilizing insulin secretagogues stimulate transcription that is directed by the cyclic adenosine 3',5'-monophosphate/calcium response element in a pancreatic islet beta-cell line.

In pancreatic beta-cells, calcium is required for insulin secretion, but can also stimulate gene transcription. High potassium-induced membrane depolarization and calcium influx have previously been shown to activate kinases that phosphorylate and thereby activate the transcription factor cAMP response element (CRE-binding protein (CREB) binding to CREs. It is unknown, however, whether hormones and neurotransmitters can activate this mechanism. Arginine vasopressin (AVP), bombesin, and acetylcholine potentiate glucose-induced insulin secretion and are known to raise cytosolic calcium levels through binding to cell surface receptors that activate phospholipase C. The effect of AVP on CRE-directed transcription was examined in the beta-cell line HIT. AVP (0.1-100 nM) stimulated gene transcription after transient transfection of a reporter gene that was placed under the transcriptional control of a CRE. This effect was inhibited by a vasopressin V1 receptor antagonist and depended on calcium influx and calcineurin phosphatase activity. By immunoblots with antiphospho-CREB antibodies and by using a Gal4-CREB fusion protein, it was shown that AVP induces the phosphorylation and activation of CREB. Like AVP, bombesin (100 nM) and the muscarinic agonist carbachol (200 microM) stimulated CRE-mediated transcription. These results show that calcium-mediating insulin secretagogues can activate CREB/CRE-directed transcription in HIT cells, offering a mechanism by which these secretagogues could produce long term effects on beta-cell function, changing the pattern of gene expression.

Animals↗
PubMed1995

Involvement of the Ca(2+)-dependent phosphatase calcineurin in gene transcription that is stimulated by cAMP through cAMP response elements.

Gene transcription can be induced by cAMP and Ca2+ through distinct protein kinases phosphorylating the transcription factor CREB, which binds to cAMP response elements (CREs) in various genes. Induction of gene transcription by Ca2+ has been shown recently to depend on the Ca2+/calmodulin-dependent protein phosphatase calcineurin in pancreatic islet cells. This study investigates the role of calcineurin in CRE-directed gene transcription after stimulation by cAMP. Reporter fusion genes under the transcriptional control of CREs were transiently transfected into the cell line HIT. Pharmacological evidence suggests that cAMP stimulates CRE-mediated transcription through a Ca(2+)-dependent mechanism. The immunosuppressive drugs cyclosporin A and FK506 inhibited CRE-mediated transcription stimulated by cAMP. At the same concentrations they also inhibited calcineurin phosphatase activity. Reversal of calcineurin inhibition by rapamycin or overexpression of calcineurin led to disinhibition of CRE-mediated gene transcription. Immunoblots with a phosphoCREB-specific antibody showed that cyclosporin A and FK506 do not interfere with CREB phosphorylation at serine 119 stimulated with cAMP or membrane depolarization. These results indicate that in HIT cells stimulation of CRE-mediated transcription depends not only on the activity of protein kinases phosphorylating CREB but also on the Ca2+/calmodulin-dependent protein phosphatase calcineurin that is necessary for the transcriptional competence of phosphorylated CREB.

Animals↗
PubMed1995

Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription.

Clinical observations agree that antidepressant drugs are effective only after a lag phase of 1-3 weeks. This delay could be explained at the molecular level by an action on gene transcription. Transcription of many genes is directed by the cAMP/Ca(2+)-responsive element (CRE) and its cognate transcription factor CRE-binding protein (CREB). Membrane depolarization and cAMP induce the phosphorylation of CREB at Ser-119 and thereby stimulate the transcriptional activity of CREB. The effect of antidepressant drugs on CREB/CRE-directed gene transcription was investigated using transient transfections of reporter fusion genes in HIT and PC-12 cells. Clomipramine, imipramine, fluoxetine, doxepin, desipramine, amitriptyline, maprotiline, mianserin, and trazodone inhibited CRE-directed gene transcription that was stimulated by membrane depolarization, with IC50 values between 70 nM and 1.73 microM. Desipramine had no effect on transcription after stimulation by cAMP but blocked the synergistic effect of cAMP and membrane depolarization to the level of stimulation by cAMP alone. Upon membrane depolarization, desipramine reduced the phosphorylation of CREB at Ser-119 and also blocked the depolarization-induced increase in the intracellular free Ca2+ concentration in HIT cells. Thus, by interfering with the depolarization-induced activation of the transcription factor CREB, antidepressant drugs can inhibit CRE-directed gene transcription, which could underlie the pharmacological effects of these clinically important drugs.

Animals↗
PubMed1993

Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization.

The cAMP-responsive element (CRE) and its cognate transcription factor CREB can mediate induction of gene transcription in response to membrane depolarization and calcium influx. In this study, the effect of cyclosporin A (CsA) and FK506 on depolarization-induced glucagon gene transcription was investigated in a pancreatic islet cell line by transfection of reporter fusion genes. CsA and FK506 inhibited depolarization-induced glucagon gene transcription, FK506 being more potent than CsA. CsA/FK506 responsiveness was mediated by the glucagon CRE and also by well characterized CREs of the choriogonadotropin and somatostatin genes. Rapamycin antagonized the inhibitory effect of FK506 but not CsA, suggesting that FK506 and CsA may act through complex formation with distinct intracellular immunophilins. Overexpression of calcineurin, which is known to be inhibited by drug-immunophilin complexes, rendered pancreatic islet cells more resistant to the inhibitory effects of CsA and FK506. These results demonstrate an inhibition by CsA and FK506 of CRE-mediated, calcium-induced transcription and suggest that membrane depolarization relies on calcineurin phosphatase activity for activation of CREB/CRE-mediated gene transcription. The interference with CRE-mediated gene transcription represents a novel mechanism of CsA/FK506 action, which may underlie pharmacological effects and toxic manifestations of these potent immunosuppressive drugs.

Activating Transcription Factor 2↗
PubMed1993

Membrane depolarization and calcium influx induce glucagon gene transcription in pancreatic islet cells through the cyclic AMP-responsive element.

Glucagon-producing pancreatic islet cells generate calcium-dependent action potentials. By the control of calcium influx through voltage-gated calcium channels, calcium is a tightly regulated second messenger in these cells. It is unknown whether calcium is a signal for glucagon gene transcription. Therefore, rat glucagon reporter fusion genes were transiently transfected into pancreatic islet cell lines. High potassium-induced membrane depolarization activated glucagon gene transcription. The effects of a calcium chelator, calcium channel blockers, calmodulin antagonists, and an inhibitor of calcium/calmodulin-dependent protein kinase II (CaM kinase II) indicate that depolarization-induced glucagon gene transcription depends on calcium influx and CaM kinase II. The depolarization-responsive element was mapped to the glucagon cAMP-responsive element (CRE). The CRE-binding protein CREB was shown, by using GAL4-CREB fusion proteins, to function as a depolarization-regulated transcription factor in pancreatic islet cells. Membrane depolarization and cAMP had synergistic effects on glucagon gene transcription. These results suggest that rat glucagon gene transcription is regulated by membrane electrical activity and calcium influx in pancreatic islet cells. This signal may be transmitted via CaM kinase II and CREB to the glucagon CRE.

Calcium↗