Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca²⁺ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺](i) by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca²⁺](i) peak followed by a sustained elevation of [Ca²⁺](i) in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min blocked the 5-HT-induced IP₃ formation and Ca²⁺ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca²⁺ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca²⁺](i) was 4 nM. Pretreatment of TSMCs with staurosporine (1 μM) of GF109203X (0.1 μM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP₃ formation and Ca²⁺ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-α, βI, βII, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC-α, βI, βII, δ, ε and θ from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-ζ was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺](i) increase or inhibit both responses independently. The translocation of PKC-α, βI, βII, δ, ε, and θ induced by PMA caused an attenuation of 5-HT-stimulated IP₃ accumulation and Ca²⁺ mobilization in TSMCs.