13 research outputs found
Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells
MAP (mitogen-activated protein) kinase (also called
Erk 1/2) plays a crucial role in cell proliferation and
differentiation. Its impact on secretory events is less
well established. The interplay of protein kinase C
(PKC), PI3-kinase nd cellular tyrosine kinase with
MAP kinase activity using inhibitors and compounds
such as glucose, phorbol 12-myristate 13-acetate
(PMA) and agonists of G-protein coupled receptors
like gastrin releasing peptide (GRP), oxytocin (OT)
and glucose-dependent insulinotropic peptide (GIP)
was investigated in INS-1 cells, an insulin secreting
cell line. MAP kinase activity was determined by
using a peptide derived from the EGF receptor as a
MAP kinase substrate and [
P
32
]ATP. Glucose as well
as GRP, OT and GIP exhibited a time-dependent
increase in MAP kinase activity with a maximum at
time point 2.5 min. All further experiments were
performed using 2.5 min incubations. The flavone PD
098059 is known to bind to the inactive forms
of MEK1 (MAPK/ERK-Kinase) thus preventing activation
by upstream activators. 20 μM PD 098059
(
IC
50
=51 μM) inhibited MAP kinase stimulated by
either glucose, GRP, OT, GIP or PMA. Inhibiton
(“downregulation”) of PKC by a long term (22h) pretreatment
with 1 μM PMA did not influence MAP
kinase activity when augmented by either of the
above mentioned compound. To investigate whether
PI3-kinase and cellular tyrosine kinase are involved
in G-protein mediated effects on MAP kinase, inhibitors
were used: 100 nM wortmannin (PI3-kinase
inhibitor) reduced the effects of GRP, OT and GIP
but not that of PMA; 100 μM genistein (tyrosine
kinase inhibitor) inhibited the stimulatory effect of
either above mentioned compound on MAP kinase
activation. Inhibition of MAP kinase by 20 μM PD
098059 did not influence insulin secretion modulated
by either compound (glucose, GRP, OT or GIP).
[
H
3
]Thymidine incorporation, however, was severely
inhibited by PD 098059. Thus MAP kinase is important
for INS-1 cell proliferation but not for its insulin
secretory response with respect to major initiators
and modulators of insulin release. The data indicate
that MAP kinase is active and under the control
of MAP kinase. PKC is upstream of a genisteinsensitive
tyrosine kinase and probably downstream
of a PI3-kinase in INS-1 cells
The FK506 binding protein 13 kDa (FKBP13) interacts with the C-chain of complement C1q
BACKGROUND: The pharmacological action of specific immunosuppressants is mediated by immunophilins. While cyclosporin A binds to cyclophilins, FK506/tacrolimus, rapamycin, and others bind to FK506 binding proteins (FKBPs). Different physiological actions of immunophilins were described but their genuine function, however, remains elusive and is still under investigation. A yeast two-hybrid screen was performed using the FK506 binding protein 13 kDa (FKBP13) as a bait and a fetal liver expression library as a prey. RESULTS: The C-chain of complement C1q (C1q-C) was detected to interact with FKBP13 in the yeast two-hybrid system and in a protein complementation assay. Neither FKBP12, FKBP25, FKBP52 nor the unrelated immunophilin CypA did react with C1q-C in the yeast system stressing the specificity of the interaction. Binding of C1q-C to FKBP13 could not be prevented in the presence of FK506, demonstrating that possibly other regions than the binding pocket of the drug are responsible for the interaction of the two proteins. CONCLUSION: It is concluded that exclusively FKBP13 but no other FKBPs tested so far interact with the C-chain of complement C1q in the two different assays and further work will be initiated to investigate the physiological relevance of the interaction
Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies
Adenosine A2B receptors of native human and rodent cell lines were investigated using [3H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [3H]PSB-298 showed saturable and reversible binding. It exhibited a KD value of 60 ± 1 nM and limited capacity (Bmax = 3.511 fmol per milligram protein) at recombinant human adenosine A2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A2B receptors. Adenosine A2B receptors were shown to be the major adenosine A2 receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [3H]PSB-298 is a selective radioligand for adenosine A2B binding sites in this cell line
Effect of Ap 4 A, UTP and Salbutamol on Mucociliary Clearance in a Mouse Model of Cystic Fibrosis (in Situ)
ABSTRACT Cystic fibrosis is a life-threatening, wide spread genetic disease diagnosed in 1 to 3000 livebirths of the Caucasian population. Here a mouse model for this disease is described and optimized using the CFTR-channel selective inhibitor CFTR(inh 172). The target parameter was mucociliary clearance measured using microdialysis of the transported fluorescent dye rhodamine in the mouse trachea in situ. The impact of Ap 4 A (diadenosine tetraphosphate) as a potential drug was investigated. Its inhalation was effective at low concentrations; established compounds such as Salbutamol and UTP increased mucociliary clearance as well. Our data show a functioning model of cystic fibrosis and the effectiveness of the newly tested Ap 4 A