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 |
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Authors: | R A John Challiss David Adams Rajendra Mistry C David Nicholson |
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Institution: | Department of Cell Physiology & Pharmacology, University of Leicester, Leicester LE1 9HN;1.NV Organon, Scientific Development Group, 53540 BH Oss, The Netherlands |
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Abstract: | - 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).
- 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 μM) concentration-dependently (−log EC50 (M), 6.55±0.15; n=3) increased cyclic AMP levels to about 70% of the maximal response to the β-adrenoceptor agonist isoprenaline.
- Rolipram per se inhibited histamine-stimulated [3H]-inositol (poly)phosphate ([3H]-InsPX) accumulation by >80% (−log EC50 (M), 6.92±0.11; n=3). Although ORG 9935 (1 μM) had little effect on histamine-stimulated [3H]-InsPX 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 [3H]-InsPX 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 μM).
- 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 μM) or ORG 9935 (1 μM) were without effect, whereas added together the inhibitors caused a significant (P<0.01) 40% reduction in the peak phasic contractile response.
- The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP3) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 μM) rolipram concentration-dependently inhibited carbachol-stimulated InsP3 accumulation by ⩾50% (−log EC50 (M), 6.77±0.21; n=4).
- Carbachol (100 μM) 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 (⩾80%) of the carbachol-stimulated InsP3 response.
- 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 InsP3 generation.
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Keywords: | Phosphodiesterases rolipram ORG 9935 cyclic AMP Ins(1 4 5)P3 phosphoinositide hydrolysis phasic contraction smooth muscle relaxation bovine tracheal smooth muscle |
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