Reduction of blood pressure elevation by losartan in spontaneously hypertensive rats through suppression of LARG expression in vascular smooth muscle cells.

BACKGROUND/PURPOSE: This study sought to elucidate the mechanism by which losartan inhibits blood pressure (BP) elevation in spontaneously hypertensive rats (SHRs). METHODS: Four-week-old Wistar-Kyoto (WKY) rats and SHRs were either treated with losartan (20 mg/kg/day) for 8 weeks or served as untreated controls. BP was measured by the tail-cuff method. At 12 weeks, isometric contraction of the aortic rings of the rats was evaluated with a force transducer and recorder. The mRNA and protein levels of the target Rho guanine nucleotide exchange factors (RhoGEFs), and the extent of myosin phosphatase target subunit 1 (MYPT-1) phosphorylation in the aorta, were determined using quantitative real-time polymerase chain reaction (qPCR) assay and Western blot analysis. RESULTS: The BP of the four-week-old SHRs did not differ from that of the age-matched WKY rats, whereas the BP of the twelve-week-old control group SHRs was higher than that of the control group WKY rats. Losartan treatment, however, inhibited BP elevation in both rat strains, doing so to a greater extent in the treatment group SHRs. The contractile force in response to angiotensin II of the aortic rings from the SHRs treated with losartan was significantly lower than that of the aortic rings from the non-treated SHRs. The protein expression of leukemia-associated RhoGEF (LARG) was significantly higher in the non-treated SHRs compared to the non-treated WKY rats. CONCLUSION: The study results showed that the reduction of BP elevation by losartan in SHRs occurs through the suppression of LARG expression and MYPT-1 phosphorylation in vascular smooth muscle cells.

Regulation on the toxicity of microcystin-LR target to protein phosphatase 1 by biotransformation pathway: effectiveness and mechanism.

Biotransformation was an important pathway to regulate the toxicity of microcystins (MCs) targeted to protein phosphatases (PPs). To explore the regulation effectiveness and mechanism, several typical biothiol transformation products originated from MCLR were prepared by nucleophilic addition reaction. The reduced inhibition effect of MCLR transformation products on PP1 was evaluated and compared with their original toxin. Though molecular simulation showed the introduced biothiols enhanced the total combination areas and energies for target complexes, the steric hindrance of introduced biothiols inhibited the combination between the key action sites (Mdha7 and Adda5 residues) and PP1. Furthermore, the introduced biothiols also weakened the hydrogen bonds for some key interaction sites and altered the ion bonds between PP1 and the two Mn2+ ions in the catalytic center. The discrepant regulation effect for biothiols on the toxicity of MCLR was closely related to above indexes and influenced by molecular sides.

Role of Protein Phosphatase 1 and Inhibitor of Protein Phosphatase 1 in Nitric Oxide-Dependent Inhibition of the DNA Damage Response in Pancreatic ß-Cells.

Nitric oxide is produced at micromolar levels by pancreatic ß-cells during exposure to proinflammatory cytokines. While classically viewed as damaging, nitric oxide also activates pathways that promote ß-cell survival. We have shown that nitric oxide, in a cell type-selective manner, inhibits the DNA damage response (DDR) and, in doing so, protects ß-cells from DNA damage-induced apoptosis. This study explores potential mechanisms by which nitric oxide inhibits DDR signaling. We show that inhibition of DDR signaling (measured by γH2AX formation and the phosphorylation of KAP1) is selective for nitric oxide, as other forms of reactive oxygen/nitrogen species do not impair DDR signaling. The kinetics and broad range of DDR substrates that are inhibited suggest that protein phosphatase activation may be one mechanism by which nitric oxide attenuates DDR signaling in ß-cells. While protein phosphatase 1 (PP1) is a primary regulator of DDR signaling and an inhibitor of PP1 (IPP1) is selectively expressed only in ß-cells, disruption of either IPP1 or PP1 does not modify the inhibitory actions of nitric oxide on DDR signaling in ß-cells. These findings support a PP1-independent mechanism by which nitric oxide selectively impairs DDR signaling and protects ß-cells from DNA damage-induced apoptosis.

High-Salt Intake Augments the Activity of the RhoA/ROCK Pathway and Reduces Intracellular Calcium in Arteries From Rats.

BACKGROUND: We investigated the influence of salt overconsumption on the functionality of the RhoA/Rho-associated kinase (ROCK) pathway and calcium regulation in arteries. METHODS: The aorta and small mesenteric arteries from rats fed a chow containing 2%, 4%, or 8% NaCl were evaluated in organ baths for the activity of the RhoA/ROCK pathway and intracellular calcium mobilization. Components of these pathways and intracellular calcium levels were also assessed in samples from 4% NaCl group. RESULTS: In arteries from animals fed regular chow, the ROCK inhibitor Y-27632 reduced the responses to phenylephrine, even when the smallest concentrations (1 and 3 µM) were tested. However, only higher concentrations of Y-27632 (10 and 50 µM) reduced phenylephrine-induced contraction in vessels from high-salt groups. Immunoblotting revealed augmented phosphorylation of the myosin phosphatase targeting subunit 1 and increased amounts of RhoA in the membrane fraction of aorta homogenates from the 4% NaCl group. Under calcium-free solution, vessels from NaCl groups presented reduced contractile responses to phenylephrine and caffeine, compared with the regular chow group. Moreover, decreased intracellular calcium at rest and after stimulation with ATP were found in aortic smooth muscle cells from 4% NaCl-fed rats, which also showed diminished levels of SERCA2 and SERCA3, but not of IP3 and ryanodine receptors, or STIM1 and Orai1 proteins. CONCLUSIONS: Arteries from rats subjected to high-salt intake are unable to properly regulate intracellular calcium levels and present augmented activity of the calcium sensitization pathway RhoA/ROCK. These changes may precede the development of vascular diseases induced by high-salt intake.

Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein Inhibitors Involve Redox-Sensitive and Signal Transducer and Activator of Transcription 3-Dependent Pathways.

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.

Amyloid-ß oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons.

Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AßOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AßOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3ß, downstream targets of CaN, prevents BDNF transport defects induced by AßOs. We further show that AßOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AßO-induced BDNF transport disruption.

High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1.

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.

Zoledronic acid in combination with serine/threonine phosphatase inhibitors induces enhanced cytotoxicity and apoptosis in hormone-refractory prostate cancer cell lines by decreasing the activities of PP1 and PP2A.

UNLABELLED: What's known on the subject? and What does the study add? Prostate cancer is the second most common cancer diagnosed among elderly men. Current standard of care with surgery, chemotherapy or radiation in prostate cancer patients are of limited efficacy, especially in the androgen refractory state of the disease, and unfortunately metastatic disease remains incurable. Skeletal metastases are the most common site for metastases for prostate cancer and bisphosphonates have been widely used for the treatment of morbidity due to skeletal related events. Zoledronic acid (ZA) is the most potent member of the nitrogen containing new generation bisphosphonate (N-BPs) family. Okadaic acid (OA) and Calyculin A (CA) are the most commonly used inhibitors of PP1 and 2A. OA, extracted from common black sponge Halachondria okaddai is a potent inhibitor of protein phosphatases, PP1 and PP2A, and CA was isolated from another marine sponge, Discodermia calyx. Therapies based on combinations of chemotherapeutics with phosphatase inhibitors that target signaling pathways within the cell with different mechanisms of action, may be useful for increasing therapeutic effect and also diminish toxic side effects by decreasing the doses of conventional chemotherapeutics. Although clinically well known, the in vitro effects of ZA on cancer cells and the underlying mechanisms are not well elucidated. In our previous studies, we have already shown anticancer effect of ZA in hormone-and drug refractory prostate cancer cells, PC-3 and DU-145. In addition to this, we have also shown that this anticancer effect may be augmented with some cytotoxic agents in prostate cancer. Now, in our present study, we have investigated whether ZA induced growth inhibition and apoptosis in PC-3 and DU-145 may be enhanced by the combination with CA or OA, through inhibition of serine/threonine phosphatases in prostate cancer cells. Both ZA/CA and ZA/OA combinations inhibited the cell viability of hormone-and drug refractory prostate cancer cells at in vivo achievable therapeutic concentrations. Moreover, a potentiation of the apoptotic effects of the combinations was also observed in the same experimental conditions. This is the first report of a synergistic combination of ZA with phosphatase inhibitors CA and OA which inhibits cell viability and induces apoptosis in human hormone and drug refractory prostate cancer cells. OBJECTIVES: • To investigate if the cytotoxic and apoptotic effect of zoledronic acid (ZA) can be enhanced by the addition of the serine/threonine protein phosphatase inhibitors calyculin A (CA) and okadaic acid (OA) in hormone and drug refractory prostate cancer cells, PC-3 and DU-145. • To discover the effect of these combination treatments on phosphatase 1 (PP1) and PP2A protein expression levels in prostate cancer cells. MATERIALS AND METHODS: • An XTT cell viability assay was used to determine cytotoxicity. • Apoptosis was evaluated by enzyme-linked immunosorbent assay (ELISA) using a Cell Death Detection ELISA Plus Kit and verified by measuring caspase 3/7 enzyme activity. • The PP1 and PP2A enzyme activities were evaluated by serine/threonine phosphatase ELISA and expression levels of PP1 and PP2A proteins were then re-assessed by Western blot analysis. RESULTS: • Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis compared with any agent alone in both PC-3 and DU-145 prostate cancer cells. • The combination of ZA with phosphatase inhibitors resulted in enhanced suppression of both PP1 and PP2A enzyme activity and protein levels, which was more overt with the ZA/CA combination. CONCLUSION: • Results from our study increase the translational potential of our in vitro findings and offer the basic rationale for the design of new combinatory strategies with ZA and phosphatase inhibitors for the treatment of prostate cancer, which may become resistant to conventional therapy.

Volatile anesthetics inhibit angiotensin II-induced vascular contraction by modulating myosin light chain phosphatase inhibiting protein, CPI-17 and regulatory subunit, MYPT1 phosphorylation.

BACKGROUND: Vascular contraction is regulated by myosin light chain (MLC) phosphorylation. Inhibition of MLC phosphatase (MLCP) increases MLC phosphorylation for a given Ca(2+) concentration, and results in promoting myofilament Ca(2+) sensitivity. MLCP activity is mainly determined by protein kinase C (PKC) and Rho kinase through the phosphorylation of both PKC-potentiated inhibitory protein (CPI-17) and myosin phosphatase target subunit (MYPT1). We have previously demonstrated that sevoflurane inhibits PKC phosphorylation and membrane translocation of Rho kinase. This study was designed to investigate the effects of sevoflurane and isoflurane on CPI-17, MYPT1, and MLC phosphorylation in response to angiotensin II (Ang II) in rat aortic smooth muscle. METHODS: The effects of sevoflurane or isoflurane (1-3 minimum alveolar concentration) on the vasoconstriction and phosphorylation of MLC, CPI-17, MYPT1 at Thr853 and MYPT1 at Thr696 in response to Ang II were investigated using isometric force transducer and Western blotting, respectively. RESULTS: Ang II (10(-7) M) elicited a transient contraction of rat aortic smooth muscle that was inhibited by both sevoflurane and isoflurane in a concentration-dependent manner. Ang II also induced an increase in the phosphorylation of MLC, CPI-17, MYPT1/Thr853 and MYPT1/Thr696. Sevoflurane inhibited the phosphorylation of MLC, CPI-17, and MYPT1/Thr853 in response to Ang II in a concentration-dependent manner. Isoflurane also inhibited MLC phosphorylation in response to Ang II, which was associated with decreases in MYPT1/Thr853, but not in CPI-17. Neither sevoflurane nor isoflurane affected the Ang II-induced phosphorylation of MYPT1/Thr696. CONCLUSION: Although both volatile anesthetics inhibited Ang II-induced vasoconstriction and MLC phosphorylation to similar extent, the mechanisms behind the inhibitory effects of each anesthetic on MLCP activity appear to differ.