Lack of effect of zaprinast on methacholine-induced contraction and inositol 1,4,5-trisphosphate accumulation in bovine tracheal smooth muscle.
1. The effects of zaprinast (M&B 22948), a selective guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterase inhibitor, and sodium nitroprusside on cyclic GMP content, phosphoinositide hydrolysis and airway smooth muscle tone were examined in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM). 2. Anion-exchange chromatography of the soluble fraction of BTSM homogenates resolved three peaks of Ca2+/calmodulin-independent phosphodiesterase (PDE) activity that corresponded to type Ia (cyclic GMP-specific, zaprinast-inhibitable), type II (cyclic GMP-stimulated) and type IV (Ro 20 1724-inhibitable) PDE isoenzymes. Zaprinast caused a selective inhibition of the type Ia PDE isoenzyme (IC50 0.94 microM) with respect to the type II and IV (IC50 s 93 microM and 197 microM respectively) isoenzymes. 3. Pretreatment of BTSM strips with zaprinast (10 microM) for 20 min affected neither the initial rate of force development, nor the resultant magnitude of contraction induced by methacholine (10 microM). In addition, zaprinast (10 microM; 20 min) did not affect the cumulative concentration-response relationship induced by methacholine. In contrast, sodium nitroprusside (300 microM) either alone, or in combination with zaprinast (10 microM), significantly attenuated tone induced by low, but not high concentrations of methacholine. This resulted in a non-parallel, rightwards shift of the methacholine concentration-response curves (nitroprusside: 4.0 fold; nitroprusside/zaprinast: 4.8 fold at the EC50 values), without a reduction in the maximum tone generated. 4. In BTSM slices, zaprinast (10 or 100 microM) did not influence basal or methacholine (10 microM)-stimulated cyclic GMP accumulation or inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation over a 60s incubation period, although it did significantly increase cyclic GMP content over longer (30 min) stimulation periods. 5. In [3H]-inositol prelabelled BTSM slices, stimulated in the presence of 5mM LiCl, methacholine (10 microM) caused a marked increase in total [3H]-inositol phosphate accumulation. This effect was not inhibited by zaprinast (10 microM), sodium nitroprusside (300 microM), or a combination of these drugs despite these agents markedly increasing tissue cyclic GMP content. 6. These findings demonstrate that despite zaprinast being a potent and selective inhibitor of the type Ia PDE isoenzyme in a cell-free system, this drug only increases cyclic GMP content in BTSM following prolonged agonist-stimulation. This may explain its lack of inhibitory effect on methacholine-induced tone. The inability of drugs which increase tissue cyclic GMP content and exhibit anti-spasmogenic activity to inhibit methacholine-stimulated Ins(1,4,5)P3 formation suggests that, unlike vascular smooth muscle, cyclic GMP-dependent mechanisms do not regulate receptor-mediated phosphoinositide hydrolysis in BTSM.
Chilvers
E R
ER
Department of Pharmacology and Therapeutics, University of Leicester.
Giembycz
M A
MA
Challiss
R A
RA
Barnes
B J
BJ
Nahorski
S R
SR
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D000818:Animals; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D005480:Flurbiprofen / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008688:Methacholine Compounds / Q000037:antagonists & inhibitors*; D009119:Muscle Contraction / Q000187:drug effects; D009129:Muscle Tonus / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D011688:Purinones / Q000494:pharmacology*; D014132:Trachea / Q000187:drug effects
D007527:Isoenzymes; D008688:Methacholine Compounds; D011688:Purinones; D009599:Nitroprusside; D005480:Flurbiprofen; D015544:Inositol 1,4,5-Trisphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C011145:zaprinast; D006152:Cyclic GMP