The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

BACKGROUNDThe phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

METHODSSeventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

RESULTSIntra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

CONCLUSIONSThe degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Adolescent ; Adult ; Aged ; Blotting, Western ; Cyclosporine ; pharmacokinetics ; therapeutic use ; Female ; Flow Cytometry ; Humans ; Immunosuppressive Agents ; pharmacokinetics ; therapeutic use ; Leukocytes, Mononuclear ; enzymology ; Liver Transplantation ; Male ; Middle Aged ; Phosphorylation ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacokinetics ; therapeutic use ; Tacrolimus ; pharmacokinetics ; therapeutic use ; Young Adult

BACKGROUNDTopical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain. The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells. The expression of c-KIT mRNA and protein of human A375 cells were also investigated.

METHODSThe cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups (10, 10(2), 10(3) and 10(4) nmol/L). The cell proliferation was measured with Cell Counting Kit-8 assays. Melanin content was measured with NaOH method. Transwell migration assay was used to measure cell migration. The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.

RESULTSThe cell proliferation of the 10(3) and 10(4) nmol/L tacrolimus groups were significantly lower (0.666 ± 0.062 and 0.496 ± 0.038) as compared with the control (0.841 ± 0.110, P < 0.05). The mean melanin content in all groups treated with different concentration of tacrolimus (10, 10(2), 10(3), 10(4) nmol/L) increased compared with the control group (P < 0.05). Dose-dependent increase in cell migration were seen in all tacrolimus-treated groups (P < 0.01). The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus (10(3) and 10(4) nmol/L) had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control (P < 0.05).

CONCLUSIONSAlthough tacrolimus had no effects on cell proliferation on A375 human melanoma cells, it could increase the melanin content and cell migration. The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control. Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.

Cell Line, Tumor ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Humans ; Immunohistochemistry ; Melanins ; metabolism ; Melanocytes ; cytology ; drug effects ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics ; Tacrolimus ; pharmacology

Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.
Active Transport, Cell Nucleus/drug effects ; *Apoptosis ; Aspirin/*pharmacology ; Cell Nucleus/*metabolism ; Humans ; MCF-7 Cells ; Phosphorylation ; Protein Binding ; Proto-Oncogene Proteins c-bcl-2/genetics/*metabolism ; Tacrolimus Binding Proteins/metabolism

The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.
Animals ; Apoptosis ; drug effects ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; Male ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; metabolism ; Spinal Cord ; drug effects ; metabolism ; pathology ; Spinal Cord Ischemia ; drug therapy ; metabolism ; Tacrolimus ; pharmacology ; therapeutic use ; Up-Regulation

OBJECTIVETo observe the change of Th immunological gene in renal transplant recipients after the treatment of cyclosporine (CsA) and tacrolimus (FK506).

METHODSThe peripheral blood lymphacytes just before and 24 hours after CsA and FK506 treatment were isolated. The total RNA of them were reverse-transcripted and examined by real-time quantity PCR array. The results were analyzed by bioinformatic methods.

RESULTSThe TLR4, CEBPB, IL4R, IL1R1,IL18R1,and IL1R2 genes were remarkably upregulated, whereas IL-2, CCL5, CD27, CCR5, CCR4, CD4, RPL13A, TGFB3, CD86, CCR3, STAT1, NFATC2IP, IL23A, IL15, IRF4, and TFCP2 were downregulated 24 hours after CsA treatment. The IL18, IL7, PTPRC, TNFSF4, SPP1, GFI1, TLR4, IL13RA1, TNF, INHBA, LAG3, IL13, IL1R1, SOCS5, IL10, YY1, TBX21, FASLG, IL18R1, and IL1R2 genes were remarkably upregulated, whereas IL-2, IL-3, IL-4, IL-6,CCR5, CD4, CD27, CD40LG, IL15, CCR3, CD86, CCR4, and IRF4 were obviously downregulated 24 hours after FK506 treatment.

CONCLUSIONCsA and FK506 exert their therapeutic effectiveness by regulating the expressions of a series of target genes.

Cyclosporine ; pharmacology ; Cytokines ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Kidney ; drug effects ; metabolism ; Kidney Transplantation ; Oligonucleotide Array Sequence Analysis ; T-Lymphocytes, Helper-Inducer ; drug effects ; metabolism ; Tacrolimus ; pharmacology

OBJECTIVETo investigate the effectiveness of pimecrolimus cream 1% for sensitive skin in adult women and its underlying mechanisms.

METHODSThe changes of subjective symptoms and signs were evaluated before and after the application of pimecrolimus cream 1% based on the severity of pruritus (SP) and severity of burning sensation (SB) scores, and on a basic syntax and molecular substrate (molecular psychophysics) of nociception and proprioception established by temperature-sensitive transient receptor potential (TRP) channels.

RESULTSThe SP and SB scores were significantly decreased in 32 patients with sensitive skin after using topical pimecrolimus cream 1% (P<0.05). Twenty (62.5%) patients showed positive capsaicin-like response (i.e. burning with consequent rapid amelioration of pruritus or burning sensation) and 6 (18.8%) showed positive camphor-like response (i.e. warming with consequent rapid amelioration of pruritus) on application sites after using the topical pimecrolimus cream 1%, and 6 (18.8%) showed negative capsaicin-like response and/or negative camphor-like response.

CONCLUSIONSPimecrolimus may rapidly inhibit or alleviate itch or burning sensation of patients with sensitive skin. The therapeutic effect of pimecrolimus is relevant to the mechanisms that activate or sensitize transient receptor potential vanilloid 1 (TRPV1) and desensitizes TRPV1 in the skin sensory afferents.

Adolescent ; Adult ; Dermatitis, Atopic ; drug therapy ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Pruritus ; drug therapy ; Skin ; drug effects ; TRPV Cation Channels ; metabolism ; Tacrolimus ; analogs & derivatives ; therapeutic use ; Young Adult

OBJECTIVETo investigate the mechanism underlying the inhibitory effect of tacrolimus (FK506) against acute liver graft rejection.

METHODSRat models of orthotopic liver transplantation were divided into 3 groups, namely the tolerance group with Brown Norway (BN) rats as the donors and Lewis rats as the recipients, rejection group with Lewis rats as donors and BN rats as recipients, and FK506 group with the same donor-recipient pair as in the rejection group and FK506 treatment. The recipients were sacrificed 7 days after the transplantation, and the hepatic histology, cytokine levels, and glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) expression in the liver and Kupffer cells were observed and detected.

RESULTSCompared with the tolerance group, the rejection group showed increased GITRL expressions in the liver and Kupffer cells (P<0.05), which was significantly lowered by FK506 treatment (P<0.05). Acute liver graft rejection caused significantly elevated interferon-γ (IFN-γ) levels and decreased interleukin-10 (IL-10) levels in the plasma and Kupffer cells (P<0.05), and these changes were obviously attenuated by FK506 treatment (P<0.05).

CONCLUSIONThe effect of FK506 in suppressing acute liver graft rejection is probably associated with down-regulated GITRL expression in the liver and Kupffer cells.

Animals ; Carrier Proteins ; metabolism ; Graft Rejection ; prevention & control ; Kupffer Cells ; metabolism ; Liver ; metabolism ; Liver Transplantation ; Male ; Rats ; Rats, Inbred BN ; Rats, Inbred Lew ; Tacrolimus ; pharmacology

OBJECTIVETo investigate the effects of tacrolimus (FK506) on behavioral function and heat-shock proteins (HSP70) expression in nervous tissues of acrylamide (ACR)-induced rats.

METHODSTotally 40 health Wistar rats were randomly divided into control group, model group, low and high doses of FK506 groups. All four groups were treated five times per week for four weeks. Gait score was measured every week. And rats were sacrificed on day 28, the cerebrum, spinal cord and sciatic nerve were dissected, and homogenized in ice bath, then the levels of HSP70 and Bcl-2, Bax were analyzed by western bloting.

RESULTSCompared with the ACR model group, the gait score in low and high doses of FK506 groups decreased by 30.1% and 47.7% respectively in the 4th week. In the cerebrum and sciatic nerve pellet, the level of HSP70 in the FK506 groups increased by 11.6%, 33.3% and 56.3%, 58.5% (P < 0.01), but no significant changes existed in spinal cord. The level of Bcl-2 in the sciatic nerve pellet increased by 39.1% (P < 0.01) but no significant changes existed in the cerebrum and spinal cord from low dose of FK506 group. And the level of Bax in the spinal cord pellet markedly increased by 46.8% but not in cerebrum and sciatic nerve pellet; Whereas in the tissues mentioned above, the levels of Bcl-2 were enhanced remarkably by 16.3%, 14.8% and 56.0% (P < 0.01) in the high dose of FK506 group. And the level of Bax in the cerebrum and spinal cord pellet markedly increased by 16.4% and 40.2% but not in sciatic nerve. The values of Bcl-2/Bax in low and high doses of FK506 groups clearly increased by 15.9%, 33.3%, 36.9% and 30.1%, 49.1%, 60.1% (P < 0.01).

CONCLUSIONThe administration of FK506 has dramatically neuroprotective effects against the development of ACR neuropathy, which may be related to up-regulating the expression of HSP70 and Bcl-2 with down-regulating the expression of Bax.

Acrylamide ; poisoning ; Animals ; HSP70 Heat-Shock Proteins ; metabolism ; Male ; Nerve Tissue ; drug effects ; metabolism ; Neuroprotective Agents ; therapeutic use ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Tacrolimus ; therapeutic use ; bcl-2-Associated X Protein ; metabolism