Abstract
In cultured canine tracheal smooth muscle cells (TSMCs), muscarinic receptor stimulation led to phosphoinositide (PI) hydrolysis. formation of inositol phosphates (IPs), and mobilization of intracellular Ca2+. Desensitization of IPs accumulation and Ca2+ mobilization evoked by carbachol was investigated using [3H]inositol labelling and Ca2+-sensitive dye fura-2. Treatment of TSMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min blocked the carbachol-stimulated formation of IPs and mobilization of Ca2+. The concentrations of PMA that gave half-maximal and maximal inhibition of carbachol-induced IPs accumulation were 70 nM and 1 μM. respectively. The inhibitory effect of PMA on carbachol-induced responses was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Treatment of TSMCs with PMA for 24 h, the cells remained the ability to response to carbachol-induced IPs accumulation and Ca2+ mobilization with the same extent as that observed in the control group. Inactive phorbol ester, 4α-phorbol 12, 13-didecanoate at 1 μM, did not inhibit the responses. The KD and Bmax of the muscarinic receptor for [3H]N-methyl scopolamine binding were not significantly changed by PMA treatment for either 30 min or 24 h. The locus of this inhibition was further investigated by examining the effect of PMA on AIF sup−inf4 -stimulated IPs accumulation in canine TSMCs. AIF sup−inf4 -induced response was inhibited by PMA treatment. supporting that G protein(s) can be directly activated by AIF sup−inf4 which was uncoupled to phospholipase C (PLC) by PMA treatment. The concomitant loss of IPs and Ca2+ mobilization is strong evidence in support of a causal relationship between PKC and IPs or Ca2+ pathways. In addition. our findings suggest that activation of PKC leads to a negative feedback regulation of carbachol-induced responses at a level distal to receptor occupancy.
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Correspondence to: Mao Yang at the above address
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Yang, C.M., Sung, TC., Ong, R. et al. Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells. Naunyn-Schmiedeberg's Arch Pharmacol 350, 77–83 (1994). https://doi.org/10.1007/BF00180014
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DOI: https://doi.org/10.1007/BF00180014