The Effect of Phorbol Ester on Airway Smooth Muscle Reactivity

Crabb, Karen Gale (1991) The Effect of Phorbol Ester on Airway Smooth Muscle Reactivity. MSc(R) thesis, University of Glasgow.

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This study was undertaken to examine the consequences of sustained activation of PKC on the reactivity of isolated rabbit and human airways. Phorbol myristate acetate (PMA), a potent phorbol ester which can substitute for DAG (Castagna et al., 1982; Yamanishi et al. ,1983), was used to directly activate PKC. The effect of incubation with PMA was examined on the contractile response to electrical field stimulation and to the endogenous agonists acetylcholine and histamine. Bronchial rings were suspended in baths containing oxygenated Krebs-Henseleit solution and changes in tension were measured isometrically. PMA (1-1007muM) potentiated cholinergic neurotransmission. The contractile response to electrical field stimulation (EFS) at 16Hz was increased in a concentration- and time-dependent manner in both human and rabbit bronchial rings, reaching a plateau at approximately 45-60 minutes. At this time point responses in the presence of PMA (10muM) were significantly greater than in the time control tissues. In the rabbit preparation both control and potentiated responses were abolished by atropine (0.1 muM). Potentiation was absent after incubation with the calcium channel blocker verapamil (10muM) and reduced after the putative PKC inhibitors H7 (10muM) and staurosporine (0.5muM). At 45 minutes into incubation, PMA (10muM but not 1muM) significantly enhanced the contractile response of rabbit bronchi to individual concentrations of ACh (0.1-30muM) within a cumulative ACh concentration response curve (0.01-300muM). There was a small leftward shift in the curve but no increase in the maximum response to ACh. The enhancement is thus of the type that would be expected of a synergism taking place beyond the receptor, rather than an increase in sensitivity of receptors. Repeating the experiment in a low calcium environment produced a rightward shift in control responses which was not seen in PMA-incubated tissues, indicating that PMA may produce an increase in the sensitivity of the preparation. Against sequential responses (at 15 minute intervals starting 45 minutes into the incubation period) to a single sub-maximal concentration of Ach (10umuM), PMA (10muM but not 1muM) significantly enhanced the first response in rabbit and human bronchi but the second and subsequent responses were not significantly greater than time controls. In the rabbit preparation the increase in response to ACh was inhibited by the calcium channel blockers nifedipine (0.3muM) and verapamil (10muM), implicating calcium influx via voltage-dependent channels in the response, and attenuated by amiloride (100muM), an inhibitor of Na+/H+ exchange. In contrast to their effects versus EFS, the protein kinase-C antagonists H7 (10muM) and staurosporine (0.5muM) did not prevent the PMA-induced potentiation of response to ACh. The cyclo-oxygenase inhibitor indomethacin (10muM) had no effect on the PMA-induced potentiation of response but itself produced a small but significant increase in response to ACh indicating an ongoing inhibitory influence of a cyclo-oxygenase product versus neurotransmission and/or contraction. The response of rabbit bronchial smooth muscle to a submaximal concentration of histamine (10muM) was also significantly increased during incubation with PMA, indicating that PMA may induce a change in intracellular coupling rather than an effect on (two sets of) receptors. Following washout in fresh Krebs second and third responses remained elevated. These results demonstrate that PMA can cause increased reactivity of rabbit and human airway smooth muscle to cholinergic nerve activation and to stimulation by ACh and histamine. They suggest that phorbol ester-induced activation of PKC is responsible, and that the potentiation of response is mediated, at least in pan, by increased fluxes of calcium and sodium ions and is independent of prostaglandin production. The findings, therefore, support the hypothesis that sustained activation of PKC in vivo could contribute to airway hyperreactivity to contractile stimuli. (Abstract shortened by ProQuest.).

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Physiology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78260
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:35
Last Modified: 30 Jan 2020 15:35

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