Modulation of spasmogen-stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle

1 The effects of isoenzyme-selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM). 2 Neither the PDE3-selective inhibitor ORG 9935, nor the PDE4-selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 mu M) concentration-dependently (-log EC50 (M), 6.55 +/- 0.15; n = 3) increased cyclic AMP levels to about 70% of the maximal response to the beta-adrenoceptor agonist isoprenaline. 3 Rolipram pe, se inhibited histamine-stimulated [H-3]-inositol (poly)phosphate ([H-3]-InsP(x)) accumulation by >80% (-log EC50 (M), 6.92 +/- 0.11; n = 3). Although ORG 9935 (1 mu M) had little effect on histamine-stimulated [H-3]-InsP(x) accumulation alone it greatly facilitated the inhibitory action of rolipram (-log EC50 (M), 8.82 +/- 0.39; n = 3). The effects of PDE3 and/or PDE4 inhibition on [H-3]-InsP(x) accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 mu M). 4 The greater-than-additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol-mediated phasic contraction, additions of rolipram (10 mu M) or ORG 9935 (1 phl) were without effect, whereas added together the inhibitors caused a significant (P<0.01) 40% reduction in the peak phasic contractile response. 5 The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP(3)) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 mu M) rolipram concentration-dependently inhibited carbachol-stimulated InsP(3) accumulation by greater than or equal to 50% (-log EC50 (M), 6.77 +/- 0.21; n = 4). 6 Carbachol (100 mu M) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca2+ from the incubation medium prevented the carbachol-evoked decrease in cyclic AMP and this coincided with a greater inhibition (greater than or equal to 80%) of the carbachol-stimulated InsP(3) response. 7 These data indicate that combined PDE3 and PDE4 inhibition has greater-than-additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor-mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP(3) generation.