The effect of okadaic acid on non‐adrenergic non‐cholinergic contraction in guinea‐pig isolated bronchus

Abstract

1 We have investigated the role of phosphatases in modulating contractile responses to electrical ®eld stimulation (EFS), methacholine, substance P and capsaicin in guinea-pig isolated main bronchus by use of the phosphatase 1 and 2A inhibitor okadaic acid. 2 Non-adrenergic non-cholinergic (eNANC) contractile responses were elicited by EFS (3 Hz, 20 s, 0.5 ms max. voltage) in the guinea-pig isolated main bronchus in the presence of the non-selective muscarinic antagonist, atropine (1 mM), the non-selective b-adrenoceptor antagonist, propranolol (1 mM), the neutral endopeptidase inhibitor thiorphan (10 mM) and the cyclo-oxygenase inhibitor, indomethacin (5 mM). Okadaic acid signi®cantly attenuated eNANC contractile responses (% inhibition) elicited by EFS (0.01 mM, 15.2+26.9%; 0.03 mM, 30.4+13.9%; 0.01 mM, 39.8+5.1%; 0.3 mM, 59.5+8.7%; 1 mM 77.8+7.8%; P50.05, n=4). In contrast, the inactive analogue 1-Nor okadaone (0.3 mM) failed to attenuate signi®cantly eNANC contractile responses (% inhibition elicited by 1-Nor okadaone, 71.25+8.5% vs dimethylsulphoxide (DMSO), 713.5+21.5%; P40.05, n=4). 3 Cholinergic contractile responses were elicited by EFS (1 ± 30 Hz, 10 s, 0.5 ms max. voltage) in guinea-pig isolated bronchus in the presence of the nitric oxide synthase inhibitor, N o -nitro-L-arginine methyl ester (L-NAME, 30 mM). Okadaic acid failed to attenuate signi®cantly the contractile (% methacholine E max ) response elicited by EFS at all frequencies tested compared with the control (1 Hz, control, 22+7.9% vs okadaic acid, 18+7.7%; 3 Hz, control, 26+6.9% vs okadaic acid, 27+9.1%; 10 Hz, control, 36+7.6% vs okadaic acid, 33+8.9%; 30 Hz, control, 50+7.6% vs okadaic acid, 42+14%; P40.05, n=4). 4 Okadaic acid (0.3 mM) failed to alter signi®cantly the contractile potency (pD 2 ) to capsaicin (okadaic acid, 9.0+0.5, vs DMSO, 9.2+0.4; P40.05 n=6), substance P (okadaic acid, 7.6+0.3 vs DMSO, 8.2+0.2; P40.05 n=7) or methacholine (okadaic acid, 6.4+0.2 vs DMSO, 6.4+0.3; P40.05 n=4). 5 Okadaic acid (0.01 ± 1 mM) did not appear to reverse substance P-induced tone. The maximal relaxant response (% reversal of substance P-induced tone) mediated by okadaic acid (1 mM) was 33+11.7% (n=4), this was not signi®cantly dierent from the DMSO (0.8%) or a time-dependent fall in tone of 34.3+23.1% (n=4) and 33+15.8% (n=4), respectively. Okadaic acid (0.3 mM) failed to augment isoprenaline-induced relaxation repsonses in substance P contracted bronchus (okadaic acid, 6.5+0.4 vs DMSO, 5.9+0.3; P40.05, n=9). 6 These results indicate that protein phosphatases appear to regulate the release of sensory neuropeptides from airway sensory nerves in response to electrical ®eld stimulation.

Stimulation of airway sensory nerves by cyclosporin A and FK506 in guinea‐pig isolated bronchus

“…Previous studies have shown that okadaic acid facilitated neuropeptide release (Hingtgen & Vasko, 1994), but had no eect on calcium entry (Cholewinski et al, 1993) or desensitization (Docherty et al, 1996) induced by capsaicin in rat DRG neurones. The latter ®ndings are consistent with a lack of eect of okadaic acid on capsaicininduced contractile responses in the guinea-pig (Harrison et al, 1997).…”

“…We have previously reported that the protein phosphatase 1 and 2A inhibitor, okadaic acid, inhibited the contractile response following stimulation of eNANC nerves but not to capsaicin in guinea‐pig isolated bronchus (Harrison et al , 1997). This suggested that protein phosphorylation/depho‐sphorylation regulates neuropeptide release from airway sensory nerves following activation of voltage‐dependent calcium channels.…”

“…We have previously demonstrated that the protein phosphatase 1 and 2A inhibitor, okadaic acid, attenuated contraction induced by stimulation of eNANC nerves in guinea‐pig isolated main bronchus (Harrison et al , 1997), suggesting that protein phosphatases regulate the release of neuropeptides from airway sensory nerves. However, the role of protein phosphatase 2B in this response is not clear.…”

The endogenous cannabinoid agonist, anandamide stimulates sensory nerves in guinea‐pig airways

“…Guinea‐pig isolated bronchus is a useful biological readout of neuropeptide release from airway sensory nerves and we have shown that various pharmacological agents including the phosphodiesterase (PDE)4 inhibitor, Ro‐201724 ( Spina et al ., 1995 ); the phosphatase 1/2A inhibitor, okadaic acid ( Harrison et al ., 1997 ) and the ORL 1 ‐receptor agonist, nociceptin ( Shah et al ., 1998 ) attenuated the eNANC response in this preparation. We have therefore investigated whether anandamide stimulates vanilloid receptors on airway sensory nerves and the role of cannabinoid receptors in modulating the eNANC response in guinea‐pig airways.…”

Force-inhibiting effect of Ser/Thr protein phosphatase 2A inhibitors on bovine ciliary muscle

“…Ref. (33)). This exceptional case might elucidate the inhibitory mechanism by comparing CCh-contraction with that with ionomycin in the BCM.…”