Modulation of agonist-stimulated second messenger and contractile events in bovine tracheal smooth muscle with cyclic nucleotide PDE inhibitors.

The aim of this thesis was to investigate and characterize the effects of isoenzyme- selective phosphodiesterase (PDE) inhibitors on a range of biochemical and functional parameters in bovine airway smooth muscle in order to further our understanding of 'cross-talk' between different second messenger pathways in this tissue.Cyclic nucleotide levels were measured after selective or non-selective PDE isoenzyme inhibition under conditions of basal and stimulated adenylyl and guanylyl cyclase activity. These data led to the conclusion that PDE IV plays an important role in the regulation of agonist-stimulated increases in cAMP levels, since under conditions of isoprenaline stimulation, incubation with the PDE IV-selective inhibitor rolipram resulted in an increase in cAMP from a control level of 20 2.7 to 50.3 4.0 pmol/mg protein. This response was potentiated in a synergistic manner by simultaneously inhibiting PDE III with ORG 9935. Co-inhibition of PDE III/IV also resulted in a significant increase in basal cAMP levels in tracheal smooth muscle slices and a marked decrease in the rolipram EC50 for cAMP accumulation in isoprenaline-stimulated slices from 205 102 to 7.3 3.0 muM. This suggested that metabolism of cAMP by PDE III is also functionally important in this tissue at least when PDE IV activity is compromized. Evidence was also obtained to suggest that cGMP is metabolized by PDE V, since incubation with zaprinast resulted in a 47% increase in basal cGMP values; however, where cGMP levels were elevated a greater increase in cGMP accumulation was seen in the presence of the nonspecific PDE inhibitor, IBMX (24.3 1.6 pmol/mg protein) compared to that seen with zaprinast (15.2 1.6 pmol/mg protein) suggesting that cGMP metabolism by other PDEs plays a significant role under these conditions. The effects of selective PDE inhibition on agonist-stimulated inositol phosphate accumulation was then investigated. Stimulation with maximally-effective concentrations of carbachol and histamine for 30 min resulted in 36- and 10-fold increases in inositol phosphate accumulations. The response to carbachol (1-100 muM) was largely unaffected by increases in cAMP accumulation caused by isoprenaline or PDE inhibition, however both manipulations inhibited the response to histamine (100muM) by approximately 80%. Again the inhibitory effects of rolipram were potentiated by ORG 9935 such that the EC50 value for rolipram-mediated inhibition was decreased from 120 27 to 1.5 0.9 muM. Such results suggest that PDE III/IV inhibition may be effective in relaxing airway smooth muscle. Consequently it was established that whilst rolipram could inhibit smooth muscle contraction stimulated by histamine or sub-maximal concentrations of methacholine, co-inhibition of PDEs with rolipram and ORG 9935 resulted in a much more potent anti-spasmogenic action. Furthermore, in contrast with the effects of rolipram alone the PDE III/IV inhibitor combination also significantly inhibited phasic contractions generated by either agonist. For example rolipram/ORG 9935 completely abolished the ability of 30uM histamine to cause a phasic contractile response. In common with previous suggestions that the membrane hyperpolarizing actions of cAMP-elevating agents may be responsible, at least in part, for the relaxation of trachealis muscle, experiments reported here suggest that PDE inhibition causes membrane hyperpolarization, possibly through increasing the open-state probability of the high- conductance, calcium-activated potassium channel, and this action may account for the mechanism whereby selective PDE inhibitors can inhibit phosphoinositide turnover and possibly as a consequence relax airway smooth muscle