Calcium/calmodulin dependent serine protein kinase (CASK), which belongs to the family of membrane associated guanylate kinase (MAGUK) proteins, has several isoforms. CASK expresses differently in embryonic tissues and adult tissues. It can be modulated by phosphorylation and SUMOylation. CASK plays an important role in neural development, spermatozoal development and renal development. Dysfunction of CASK may lead to diseases. CASK is distributed extensively in the brain, regulating synapse formation. Mutation of CASK can lead to several neurologic diseases. CASK is also involved in the development and maturation of sperm and fertilization. It also can influence renal development through interaction with DLG1.
Animals ; Disease ; genetics ; Embryonic Development ; Guanylate Kinases ; genetics ; Humans

OBJECTIVETo investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell.

METHODS(1) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4.1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)], blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 µg protein), CASK antibody precipitation group (100 µg protein), IgG antibody group (100 µg protein), Western blot group (20 µg protein). Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn), KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/L PBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group (transfected with pcDNA-Achn vector), and blank control group (treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10.777, 6.112, P values all below 0.05). The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3.1 group (with t value respectively 5.367, 6.053, 9.831, P values all below 0.05). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group (with t value respectively 5.481, 9.517, P values all below 0.05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [(15.6 ± 0.5)%] as compared with that in LPS induction group [(32.8 ± 2.6)%, t = 10.083, P < 0.05], and that in cotransfection group showed further inhibition [(7.0 ± 2.0)%, t = 9.827, P < 0.01]. (4) Apoptosis rate in Achn inhibition group [(45.6 ± 10.9)%] was higher than that in blank control group [(13.2 ± 4.3) %, t = 7.043, P < 0.05]; while that in Achn induction group [(5.3 ± 2.9)%] was lower than that in blank control group (t = 6.499, P < 0.05).

CONCLUSIONSAchn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.

Apoptosis ; Autoantigens ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Endothelial Cells ; cytology ; Guanylate Kinases ; metabolism ; Humans ; Ribonucleoproteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the expression of calcium/calmodulin-dependent serine protein kinase (CASK) induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event.

METHODSEA. hy926 cells were cultured in normoxic condition for 0, 12, 24, 48, 72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1, 3, 6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3, 6, 12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 (0, 10, 100 nmol/L and 1,10 micromol/L) 1h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot.

RESULTSCASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia (0.010 +/- 0.003, P < 0.01), and it reached the peak 12 hrs after hypoxia (0.192 +/- 0.023, P < 0.01). The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 micromol/L SP600125 pretreatment.

CONCLUSIONJNK signal pathway is involved in short-term hypoxia related CASK upregulation.

Calcium ; metabolism ; Cell Hypoxia ; Cell Line ; Endothelial Cells ; metabolism ; physiology ; Guanylate Kinases ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Signal Transduction

Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (K(Ca)) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated K(Ca) currents were blocked by iberiotoxin (IbTX, a large conductance K(Ca) blocker). The H2O2-stimulating effect on large-conductance K(Ca) (BK(Ca)) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BK(Ca) currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BK(Ca) currents. Using RT-PCR and western blot analysis, three subtypes of K(Ca) channels were detected in HCFs: BK(Ca) channels, small-conductance K(Ca) (SK(Ca)) channels, and intermediate-conductance K(Ca) (IK(Ca)) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BK(Ca) currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BK(Ca) channels.
Apoptosis ; Blotting, Western ; Cyclic AMP-Dependent Protein Kinases ; Cyclic GMP-Dependent Protein Kinases ; Fibroblasts* ; Guanosine ; Guanylate Cyclase ; Humans* ; Hydrogen Peroxide* ; Hydrogen* ; Oxidative Stress ; Phosphotransferases ; Potassium Channels, Calcium-Activated ; Protein Kinases*

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) reduces the intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells, whereas the effect of NO on [Ca2+]i in endothelial cells is still controversial. Therefore, the effect of NO on the [Ca2+]i, and its mechanism in mouse aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) were examined. MATERIALS AND METHODS: In primary cultured MAEC and HUVEC, cells were loaded with fura 2-AM and [Ca2+]i and measured using a microfluorometer. RESULTS: The NO donor, sodium nitroprusside (SNP), reduced the [Ca2+]i in 72% of the cells tested (n=100). In the remaining cells, the effect of SNP was biphasic, or the [Ca2+]i was increased. In addition, the membrane-permeable cGMP, 8-bromo cGMP, decreased the [Ca2+]i. The effects of SNP and 8-bromo cGMP were inhibited by the soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and the cGMP-dependent protein kinase inhibitor, KT5823, respectively. In contrast, in the presence of 8-bromo cGMP or ODQ, SNP increased the [Ca2+]i. CONCLUSION: These results suggest that NO inhibits the [Ca2+]i through a cGMP-dependent mechanism and increases the [Ca2+]i through a cGMP-independent mechanism.
Animals ; Cyclic GMP ; Endothelial Cells* ; Endothelium ; Guanylate Cyclase ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Myocytes, Smooth Muscle ; Nitric Oxide ; Nitroprusside ; Protein Kinases ; Tissue Donors

This study was designed to elucidate high K(+)-induced relaxation in the human gastric fundus. Circular smooth muscle from the human gastric fundus greater curvature showed stretch-dependent high K+ (50 mM)-induced contractions. However, longitudinal smooth muscle produced stretch-dependent high K(+)-induced relaxation. We investigated several relaxation mechanisms to understand the reason for the discrepancy. Protein kinase inhibitors such as KT 5823 (1 microM) and KT 5720 (1 microM) which block protein kinases (PKG and PKA) had no effect on high K(+)-induced relaxation. K+ channel blockers except 4-aminopyridine (4-AP), a voltage-dependent K+ channel (KV) blocker, did not affect high K(+)-induced relaxation. However, N(G)-nitro-L-arginine and 1H-(1,2,4)oxadiazolo (4,3-A)quinoxalin-1-one, an inhibitors of soluble guanylate cyclase (sGC) and 4-AP inhibited relaxation and reversed relaxation to contraction. High K(+)-induced relaxation of the human gastric fundus was observed only in the longitudinal muscles from the greater curvature. These data suggest that the longitudinal muscle of the human gastric fundus greater curvature produced high K(+)-induced relaxation that was activated by the nitric oxide/sGC pathway through a KV channel-dependent mechanism.
4-Aminopyridine ; Carbazoles ; Contracts ; Gastric Fundus ; Guanylate Cyclase ; Humans ; Muscle, Smooth ; Muscles ; Nitric Oxide ; Protein Kinase Inhibitors ; Protein Kinases ; Pyrroles ; Relaxation

This study was designed to elucidate high-K+induced response of circular and longitudinal smooth muscle from human gastric corpus using isometric contraction. Contraction from circular and longitudinal muscle stripes of gastric corpus greater curvature and lesser curvature were compared. Circular smooth muscle from corpus greater curvature showed high K+ (50 mM)-induced tonic contraction. On the contrary, however, longitudinal smooth muscle strips showed high K+ (50 mM)-induced sustained relaxation. To find out the reason for the discrepancy we tested several relaxation mechanisms. Protein kinase blockers like KT5720, PKA inhibitor, and KT5823, PKG inhibitor, did not affect high K+-induced relaxation. K+ channel blockers like tetraethylammonium (TEA), apamin (APA), glibenclamide (Glib) and barium (Ba2+) also had no effect. However, N(G)-nitro-L-arginine (L-NNA) and 1H-(1,2,4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC) and 4-AP (4-aminopyridine), voltage-dependent K+ channel (KV) blocker, inhibited high K+-induced relaxation, hence reversing to tonic contraction. High K+-induced relaxation was observed in gastric corpus of human stomach, but only in the longitudinal muscles from greater curvature not lesser curvature. L-NNA, ODQ and KV channel blocker sensitive high K+-induced relaxation in longitudinal muscle of higher portion of corpus was also observed. These results suggest that longitudinal smooth muscle from greater curvature of gastric corpus produced high K+-induced relaxation which was activated by NO/sGC pathway and by KV channel dependent mechanism.
Apamin ; Barium ; Carbazoles ; Contracts ; Glyburide ; Guanylate Cyclase ; Humans ; Intracellular Signaling Peptides and Proteins ; Isometric Contraction ; Muscle, Smooth ; Muscles ; Protein Kinases ; Pyrroles ; Relaxation ; Stomach ; Tetraethylammonium

The serine/threonine kinase Akt has been shown to play a role of multiple cellular signaling pathways and act as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI3K). It has been reported that phosphorylated Akt activates eNOS resulting in the production of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). It has been also reported that PKG activates PI3K/Akt signaling. Therefore, it is possible that PI3K, Akt, eNOS, sGC, and PKG form a loop to exert enhanced and sustained activation of Akt. However, the existence of this loop in eNOS-expressing cells, such as endothelial cells or astrocytes, has not been reported. Thus, we examined a possibility that Akt phosphorylation might be enhanced via eNOS/sGC/PKG/PI3K pathway in astrocytes in vivo and in vitro. Phosphorylation of Akt was detected in astrocytes after KA treatment and was maintained up to 72 h in mouse hippocampus. 2 weeks after KA treatment, astrocytic Akt phosphorylation was normalized to control. The inhibition of eNOS, sGC, and PKG significantly decreased Akt and eNOS phosphorylation induced by KA in astrocytes. In contrast, the decreased phosphorylation of Akt and eNOS by eNOS inhibition was significantly reversed with PKG activation. The above findings in mouse hippocampus were also observed in primary astrocytes. These data suggest that Akt/eNOS/sGC/PKG/PI3K pathway may constitute a loop, resulting in enhanced and sustained Akt activation in astrocytes.
Animals ; Astrocytes ; Cyclic GMP-Dependent Protein Kinases ; Endothelial Cells ; Guanylate Cyclase ; Hippocampus ; Kainic Acid ; Mice ; Nitric Oxide ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Phosphotransferases ; Receptors, Growth Factor ; Transducers

OBJECTIVETo verify whether abnormal expression of calcium/calmodulin dependent serine protein kinase (CASK) and inhibitors of differentiation 1 (ID1) exist in Fb of keloid, and to observe the effect of artesunate on two genes.

METHODSFifteen samples of keloid and 12 samples of normal skin tissue (discarded) excised from patients admitted to our hospital were collected. Tissue particle adherent method was used in the primary culture of Fb, and cells from the third to the eighth passage were used for test. Expressions of CASK and ID1 in Fb harvested from both sources were observed with immunofluorescence staining. Fb of keloid were stimulated with artesunate in various concentration for different time, and the median inhibitory concentration (IC50) was determined with the MTT colorimetric assay, which served as the intervention concentration of artesunate. Fb of normal skin were set as normal control group (NC, treated with medium solution). Fb of keloid were divided into scar control group (SC, treated with medium solution) and scar administration group (SA, treated with artesunate in IC50). The cycle and apoptosis of Fb were detected with flow cytometric assay, and the nucleic acid and protein expressions of CASK and ID1 of Fb in each group were determined with RT-PCR and Western blotting. Data were processed with one-way analysis of variance and LSD-t test.

RESULTSExpressions of CASK and ID1 were detected in two kinds of Fb. The concentration of 75 mg/L was selected as the intervention concentration of artesunate. (1) There were statistically significant differences among the three groups in the percentages of cells in G0/G1 phase and G2/M phase (with F values respectively 118.064 and 163.840, P values all below 0.01). The percentage of cells in G0/G1 phase of group SA was (91.4 ± 1.4)%, which was significantly higher than that of group SC and group NC [respectively (80.7 ± 0.3)% and (82.4 ± 0.6)%, with t values respectively 12.740 and 9.872, P values all below 0.05]. The percentage of cells in G2/M phase of group SA was (6.9 ± 0.3)%, which was significantly lower than that of group SC and group NC [respectively (13.7 ± 0.3)% and (12.7 ± 0.8)%, with t values respectively 43.702 and 12.276, P values all below 0.05]. (2) There were statistically significant differences among the three groups in the early and late apoptotic rates (with F values respectively 61.879 and 4710.862, P values all below 0.01). The early and late apoptotic rates of group SA were respectively (7.1 ± 1.0)% and (14.9 ± 0.3)%, which were significantly higher than those of group SC and group NC [with early apoptotic rate respectively (2.6 ± 0.4)% and (2.7 ± 0.3)%, t values respectively 7.974 and 7.767, P values all below 0.05; with late apoptotic rate respectively (2.3 ± 0.3)% and (2.5 ± 0.4)%, t values respectively 72.882 and 69.792, P values all below 0.05]. (3) The mRNA expression of CASK in group SC was 0.658 ± 0.024, and it was lower than that of group NC (1.076 ± 0.008, t = 28.997, P < 0.01) and group SA (0.855 ± 0.008, t = 13.549, P < 0.01). The protein expression of CASK in group SC was 0.067 ± 0.007, and it was lower than that of group NC (0.179 ± 0.015, t = 12.042, P < 0.01) and group SA (0.132 ± 0.010, t = 9.498, P < 0.01). (4) The mRNA expression of ID1 in group SC was 0.416 ± 0.006, which was higher than that of group NC (0.317 ± 0.020, t = 8.299, P < 0.01) and group SA (0.217 ± 0.009, t = 32.417, P < 0.01). The protein expression of ID1 in group SC was 0.789 ± 0.034, and it was higher than that of group NC (0.366 ± 0.029, t = 16.341, P < 0.01) and group SA (0.114 ± 0.006, t = 33.978, P < 0.01).

CONCLUSIONSIt is speculated that CASK and ID1 participate in the proliferation of Fb in keloid. The mechanism of artesunate in inhibiting the proliferation of Fb in keloid may be related to the up-regulation of CASK and down-regulation of ID1.

Adolescent ; Adult ; Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Fibroblasts ; metabolism ; Gene Expression Regulation ; Guanylate Kinases ; genetics ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; Male ; Middle Aged ; Young Adult

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents (Icap). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on Icap. Membrane-permeable cGMP analogs, 8-bromoguanosine 3', 5'-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on Icap. The PKG inhibitor KT5823 prevented the inhibition of Icap by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.
Animals ; Benzoates ; Carbazoles ; Cyclic GMP-Dependent Protein Kinases ; Ganglia, Spinal ; Guanosine ; Guanylate Cyclase ; Hemoglobins ; Humans ; Imidazoles ; Neurons ; Nitric Oxide ; Nitroprusside ; Penicillamine ; Phosphotransferases ; Proteins ; Rats ; Receptors, Cytoplasmic and Nuclear ; Sensory Receptor Cells ; Spinal Nerve Roots ; Tissue Donors