tacrolimus and Hypoxia

A-kinase anchoring protein 5 (AKAP5) is involved in ventricular remodeling in rats with heart failure after myocardial infarction; however, the specific mechanism is not clear. This study investigated whether AKAP5 anchors calcineurin (CaN) to regulate the remodeling of H9c2 cardiomyocytes.. H9c2 cells were subjected to hypoxia stress for 3 h and reoxygenation for 24 h to create a hypoxia-reoxygenation (H/R) model. These cells were divided into three groups: H/R (model), empty vector +H/R (NC), and siRNA-AKAP5+H/R (siRNA-AKAP5) groups. The non-H/R H9c2 cells were used as normal controls. Western blotting was used to detect cardiac hypertrophy-related protein expression in the cells, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta myosin heavy chain (β-MHC), and phosphorylated nuclear factor of activated T-cell 3 (p-NFATc3). Phalloidin staining was used to label the cytoskeleton and the cell area in different groups was measured. Immunofluorescence staining and coimmunoprecipitation were used to study the relationship between AKAP5 and CaN. H9c2 cells pretreated with the CaN inhibitor FK506 were used to further verify the relationship between AKAP5 and CaN.. In the siRNA-AKAP5+H/R group, the expression level of cardiac hypertrophy-related proteins (ANP, BNP, and β-MHC) and CaN and the area of cardiomyocytes were significantly increased, while the p-NFATc3/NFATc3 ratio was decreased in H9c2H/R cells. AKAP5 and CaN proteins were colocalized and interacted in the cells. The CaN inhibitor significantly suppressed the expression of CaN, increased the p-NFATc3/NFATc3 ratio, and reduced the expression levels of ANP, BNP, and β-MHC proteins in the cells with low AKAP5 expression.. AKAP5 downregulation aggravated the remodeling of cardiomyocytes after H/R. AKAP5 may anchor CaN to form a complex, which in turn activates NFATc3 dephosphorylation and expression of hypertrophy-related proteins.

Topics: A Kinase Anchor Proteins; Animals; Atrial Natriuretic Factor; Calcineurin; Cardiomegaly; Hypoxia; Myocytes, Cardiac; Myosin Heavy Chains; Natriuretic Peptide, Brain; Phalloidine; Rats; RNA, Small Interfering; Tacrolimus

FK-506 (Tacrolimus) is a very commonly used immunomodulatory agent that plays important roles in modulating the calcium-dependent phosphoserine-phosphothreonine protein phosphatase calcineurin and thus inhibits calcineurin-mediated secondary neuronal damage. The biological function of FK-506 in the spinal cord has not been fully elucidated. To clarify the anti-inflammatory action of FK-506 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats and hypoxia-treated primary spinal cord microglia cultures. This work studied the activation of NF-κB and proinflammatory cytokine (TNF-a, IL-1b, and IL-6) expression. ELISA and q-PCR analysis revealed that TNF-a, IL-1b, and IL-6 levels significantly increased 3 days after spinal cord contusion and decreased after 14 days, accompanied by the increased activation of NF-κB. This increase was reversed by an FK-506 treatment. Double immunofluorescence labeling suggested that NF-κB activation was especially prominent in microglia. Immunohistochemistry confirmed no alteration in the number of microglia. Moreover, the results in hypoxia-treated primary spinal cord microglia confirmed the effect of FK-506 on TNF-a, IL-1b, and IL-6 expression and NF-κB activation. These findings suggest that FK-506 may be involved in microglial activation after SCI.

Topics: Animals; Apoptosis; CD11b Antigen; Cell Proliferation; Cells, Cultured; Cytokines; Hypoxia; Inflammation; Inflammation Mediators; Male; Microglia; NF-kappa B; Phosphorylation; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Tacrolimus

Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes.. Astrocytes, the most abundant cell type in the brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission.

Topics: Action Potentials; Animals; Astrocytes; Calcium Channel Blockers; Calcium Signaling; Enzyme Inhibitors; GAP-43 Protein; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Hypoxia; In Vitro Techniques; Microtubule-Associated Proteins; Mitochondria; Organ Culture Techniques; Rats; Rats, Transgenic; Sodium Channel Blockers; Tacrolimus; Tetrodotoxin; Time Factors

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca(2+) concentration ([Ca(2+)]i). Recent studies suggest that hydrogen sulfide (H2S) may serve as an oxygen sensor/signal in the carotid body during acute hypoxia. To further test such a role for H2S, we studied the effects of H2S on the activity of TASK channel and [Ca(2+)]i, which are considered important for mediating the glomus cell response to hypoxia. Like hypoxia, NaHS (a H2S donor) inhibited TASK activity and elevated [Ca(2+)]i. To inhibit the production of H2S, glomus cells were incubated (3h) with inhibitors of cystathionine-β-synthase and cystathionine-γ-lyase (DL-propargylglycine, aminooxyacetic acid, β-cyano-L-alanine; 0.3 mM). SF7 fluorescence was used to assess the level of H2S production. The inhibitors blocked L-cysteine- and hypoxia-induced elevation of SF7 fluorescence intensity. In cells treated with the inhibitors, hypoxia produced an inhibition of TASK activity and a rise in [Ca(2+)]i, similar in magnitude to those observed in control cells. L-cysteine produced no effect on TASK activity or [Ca(2+)]i and did not affect hypoxia-induced inhibition of TASK and elevation of [Ca(2+)]i. These findings suggest that under normal conditions, H2S is not a major signal in hypoxia-induced modulation of TASK channels and [Ca(2+)]i in isolated glomus cells.

Topics: Alkynes; Analysis of Variance; Animals; Animals, Newborn; Calcium; Carotid Body; Chemoreceptor Cells; Dose-Response Relationship, Drug; Gasotransmitters; Glycine; Hydrogen Sulfide; Hypoxia; Intracellular Fluid; Membrane Potentials; Nerve Tissue Proteins; Patch-Clamp Techniques; Potassium Channels, Tandem Pore Domain; Rats; Rats, Sprague-Dawley; Sodium Compounds; Tacrolimus; Time Factors

Pancreatic islet transplantation is a promising method for curing diabetes mellitus. We proposed in this study a molecularly engineered islet cell clusters (ICCs) that could overcome problems posed by islet transplantation circumstances and host's immune reactions. A gene containing highly releasable exendin-4, an insulinotropic protein, was delivered into single islet cells to enhance glucose sensitivity; thereafter, the cells were reaggregated into small size ICCs. Then the surface of ICCs was modified with biocompatible poly(ethylene glycol)-lipid (PEG) (C18) for preventing immune reactions. The regimen of ICCs with low doses of anti-CD154 mAb and tacrolimus could effectively maintain the normal glucose level in diabetic mice. This molecularly engineered PEG-Sp-Ex-4 ICC regimen prevented cell death in transplantation site, partly through improving the regulation of glucose metabolism and by preventing hypoxia- and immune response-induced apoptosis. Application of this remedy is also potentially far-reaching; one would be to help overcome islet supply shortage due to the limited availability of pancreas donors and reduce the immunosuppressant regimens to eliminate their adverse effects.

Topics: Animals; Antibodies; Apoptosis; CD40 Ligand; Cell Survival; Cells, Cultured; Diabetes Mellitus, Experimental; Exenatide; Genetic Engineering; Glucose; Hypoxia; Islets of Langerhans; Islets of Langerhans Transplantation; Mice; Mice, Inbred C57BL; Mice, Nude; Peptides; Polyethylene Glycols; Tacrolimus; Venoms

We investigated clinical factors that affected the clearance of tacrolimus (FK506) administered by continuous drip infusion to children who had received allogeneic hematopoietic SCT. Blood FK506 levels were measured every day in 27 patients in an attempt to adjust the dose to maintain the target range (10-15 ng/mL). Patients who developed engraftment syndrome (ES) and acute GVHD and patients less than 7 years of age showed a higher FK506 clearance calculated from body weight (BW) for 5 or more consecutive days compared with the control groups. A time-course study showed that the occurrence of ES, but not acute GVHD, was related to increased clearance of FK506. When calculated from body surface area (BSA), a significant increase in FK506 clearance was observed in patients with ES, but not in those less than 7 years of age. FK506 clearance was not influenced by CYP3A5, multidrug resistance 1 or ABCG2 genotypes. None of the clinical parameters affected blood FK506 levels. Determination of the FK506 dose on the basis of frequent monitoring of the blood concentration seems to minimize the serious adverse effects induced by the immunosuppressant. It may be more accurate to dose FK506 according to BSA rather than BW for pediatric patients.

Topics: Adolescent; Aging; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Child; Child, Preschool; Cytochrome P-450 CYP3A; Drug Dosage Calculations; Erythema; Female; Fever; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Hypoxia; Immunosuppressive Agents; Infant; Male; Metabolic Clearance Rate; Neoplasm Proteins; Polymorphism, Genetic; Pulmonary Eosinophilia; Syndrome; Tacrolimus; Weight Gain

Hypoxia is the most common cause of perinatal seizures and can be refractory to conventional anticonvulsant drugs, suggesting an age-specific form of epileptogenesis. A model of hypoxia-induced seizures in immature rats reveals that seizures result in immediate activation of the phosphatase calcineurin (CaN) in area CA1 of hippocampus. After seizures, CA1 pyramidal neurons exhibit a downregulation of GABA(A) receptor (GABA(A)R)-mediated inhibition that was reversed by CaN inhibitors. CaN activation appears to be dependent on seizure-induced activation of Ca2+-permeable AMPA receptors (AMPARs), because the upregulation of CaN activation and GABA(A)R inhibition were attenuated by GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] or Joro spider toxin. GABA(A)R beta2/3 subunit protein was dephosphorylated at 1 h after seizures, suggesting this subunit as a possible substrate of CaN in this model. Finally, in vivo administration of the CaN inhibitor FK-506 significantly suppressed hypoxic seizures, and posttreatment with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) or FK-506 blocked the hypoxic seizure-induced increase in CaN expression. These data suggest that Ca2+-permeable AMPARs and CaN regulate inhibitory synaptic transmission in a novel plasticity pathway that may play a role in epileptogenesis in the immature brain.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Blotting, Western; Calcineurin; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Hippocampus; Hypoxia; Immunoprecipitation; In Vitro Techniques; Neural Inhibition; Patch-Clamp Techniques; Rats; Receptors, AMPA; Receptors, GABA-A; Seizures; Synapses; Synaptic Transmission; Tacrolimus; Time Factors

Reductions in hepatic oxygen supply may reduce the oxidative metabolism of drugs, including tacrolimus. We encountered a patient (2.3-year-old girl) with hypoxemia [arterial oxygen tension (PaO2) 40.9 mmHg in room air] due to hepatopulmonary syndrome who had undergone living related liver transplantation. After transplantation, tacrolimus was initially administered by continuous intravenous infusion, and her PaO2 was maintained at more than 50 mmHg [72.8+/-10.4 (SD) mmHg] by oxygen supplementation. Apparent clearance of tacrolimus (calculated as: the infusion rate of tacrolimus/blood concentration) in the patient (0.075 l/h per kg) was comparable to those of non-hypoxemic control pediatric cases (0.092+/-0.014 l/h per kg, n=7, mean age 2.2 years, PaO2 149.2+/-41.5 mmHg), except for the acute decline in the early period after transplantation. These findings suggest that the reduction in tacrolimus clearance is negligible when arterial oxygen tension is maintained at more than 50 mmHg, even in patients with hypoxemia.

Topics: Arteries; Case-Control Studies; Child, Preschool; Female; Hepatopulmonary Syndrome; Humans; Hypoxia; Immunosuppressive Agents; Infant; Infusions, Intravenous; Liver Transplantation; Male; Oxygen; Partial Pressure; Postoperative Period; Tacrolimus