| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 86 === Summary
Recent studies have shown that phosphatidylinositol 3-kinase
(PI3-K), protein phosphatase (e.g. calcineurin) play a
role in regulating cellular growth, Ca2+-channel and
glucose uptake. It is considered that they may be
involved in modulating the transmitter release from the motor
nerve terminals as well as in the skeletal muscle
contractions. Since it has not been reported, we thus
attempted to aim at exploring the possible roles of PI3-K
and calcineurin in the isolated mouse phrenic nerve-diaphragm
preparation.
Our model system for testing this working hypothesis is that
acetylcholine (ACh) release from the motor nerve terminals is
monitored by either the differentiation of muscle
contractions evoked by nerve stimulation from those
by direct muscle stimulation or by
electrophysiological measurement of end plate potentials. The
known compounds used to enhance ACh release are K+-
channel blockers (UO22+, 4-AP and TEA) and that to
inhibit ACh release is beta-bungarotoxin (beta- BuTx). The
specific inhibitor of PI3-K (wortmannin) and that of
calcineurin (cyclosporine) were adopted as the tools for this
purpose in exploring their interaction with the known
compounds in altering ACh release and from these
results obtained to delineate the possible role of PI3-K
and calcineurin involved in ACh release.
In order to test whether cyclosporine affects the skeletal
muscle contractions. We studied its effects on the
potentiating effects of Na+- channel activator
(deltamethrine and veratridine) as well as Cl--channel
blockers (9-AC and Cd2+). The results obtained might shed some
light on the possible roles of calcineurin in skeletal
muscle contractions.
Effects of wortmannin (WM) on ACh release
UO22+ exerted a marked potentiating effect on the muscle
contractions evoked by nerve stimulation, which could
be divided into two phases (phase I and phase II).
This potentiated contractions could be further enhanced
by TEA. WM pretreatment dramatically enhanced phase II
contraction accompanied with muscle fasciculation which could be
abolished by d-TC, indicating that the observed contraction
potentiation was neurogenic rather myogenic. The
specificity of this effect by WM was evident as none of
the phase I of UO22+ -potentiated contractions, the
potentiating effects of TEA or beta-BuTx administered in vivo
abolished the potentiation by WM.
This result suggests a possibility that the site of action of WM
is similar to that of UO22+ , perhaps a K+-channels.
This finding implied that PI3-K may involve in activating
K+-channel on the motor nerve terminals while
calcineurin activation favored K+-channel inactivation.
Effects of cyclosporine on diaphragm muscle contractions
Deltamethrine (DTM), a Na+-channel activator, markedly
potentiated the myogenic contractions, which could
be abolished by either cyclosporine A (CsA) or
beta-BuTx administered in vivo which may induce
spreading of AChR in the non-junctional region. Combined d-TC
with PDBu but not PDBu alone completely reversed the inhibitory
effect of CsA on DTM. This finding suggests that
calcineurin inhibition by CsA caused ion channels in
phosphorylated form, resulting in membrane
hyperpolarization which blocked the activation of Na+-channel by
DTM. This speculation is supported by the finding that
phosphorylation of Cl-- channel by activation of PKC with
PDBu (as previously proofed) restored the membrane
excitability combined with the blockade of nonselective
cation channel of AChR by d-TC completely prevented the
inhibition by CsA.
In concert with this finding, the inhibitory effect of CsA on
veratridine-induced muslce contraction was augmented by d-TC,
suggesting that AChR-channels are cooperated with the membrane
ion channels in regulating the ion channel
activities and the membrane potentials. PKC and
calcineurin in the skeletal muscle may participate in
modulating ion channel activity by modifying protein
phosphorylation and dephosphorylation.
In conclusion, we have first demonstrated in this study that
PI3-K and calcineurin may involve in regulating K+
channels on motor nerve terminals while PKC and
calcineurin in the skeletal muscle exhibit their
regulatory effects on the ion channels (Na+ and Cl-) through
protein phosphorylation and dephosphorylation
pathway.
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