| Summary: | 碩士 === 輔仁大學 === 生物學研究所 === 83 === Regulation of activation of protein kinase C (PKC) by phorbol
12-myristate 13-acetate (PMA) and protein kinase A (PKA) by
cyclic adenosine 3',5'-monophosphate elevating agent, forskolin
(an activator of adenylate cyclase) on carbachol-induced
increase in intracellular Ca2+ and phosphoinositide (PI)
hydrolysis was investigated in cultured canine tracheal smooth
muscle cells. PMA and forskolin inhibited the carbachol-induced
responses in a dose-dependent manner with maximal inhibition at
concentration of 1 μM and 100 μM, respectively, but had no
effect on the basal level; PMA and forskolin produced
depression of the maximal responses and a shift to the right in
the dose-effect curves. Staurosporine (an inhibitor of PKC) and
HA-1004 (an inhibitor of PKA) blocked the inhibitory effects of
PMA and forskolin on carbachol-induced IPs accumulation,
respectively. The inhibitory effect of PMA was reduced during
prolonged treatment for more than 4 h and blocked after 24 h
treatment. PMA induced increase in membrane-bound PKC activity
at 30 min, decreased after 2 h and persisted for at least 24 h
treatment; PMA caused decrease of the total PKC activity with
exposure longer than 2 h. These results suggested that the
inhibitory effects of PMA and forskolin on carbachol-induced
IPs accumulation were mediated through activation of PKC and
PKA, respectively. The Kd and Bmax of the muscarinic receptor
were not changed by PMA and forskolin treatment, suggesting the
negative feedback regulation of PKC and PKA at a level distal
to receptor. The inhibitory effects of PMA and forskolin on IPs
accumulation induced by fluoroaluminate supported the target
site of PKC and PKA might be at G proteins. Taken together,
activation of PKC and PKA may inhibit PI hydrolysis by altering
the function of G proteins and then affect Ca2+ mobilization.
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