Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl- currents by NO donors in cat tracheal smooth muscle

Abstract

1. The effects of NO donors on Ca2+-dependent Cl- currents (I(Cl(Ca))) were investigated in freshly isolated cat tracheal myocytes using the whole-cell patch clamp technique. 2. With nystatin-perforated whole-cell recording, carbachol (CCh, ≤ 1 μM) induced a transient inward current (I(CCh)) with a reversal potential of about -20 mV. Activation of I(CCh) probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) greatly inhibited this current, while 11-(2-(diethlylamino)methyl)-1-piperidinylacetyl)-5,11-dihydro-6H-pyrido (2,3β) (1,4)benzodiazepine-6-one (AF-DX 116) or pirenzepine at concentrations of up to 1 μM were almost ineffective. 3. Chloride channel/transporter blockers such as DIDS (100 μM), anthracene-9-carboxylic acid (9-AC, 100 μM) and niflumic acid (100 μM) greatly inhibited I(CCh), but cation channel blockers, such as nifedipine (10 μM), Zn2+ (500 μM) or Gd3+ (500 μM), were without effect. 4. Activation of I(CCh) was strongly attenuated by pretreatment with ryanodine (4 μM) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml-1) in the pipette abolished a substantial part of I(CCh). These results suggest that I(CCh) is I(Cl(Ca)), which is activated by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. 5. The nitric oxide donor S-nitroso-N-acetyl penicillamine (SNAP) reduced the amplitude of I(CCh) dose dependently (IC50 ~ 10 μM). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (±)-E-methyl-2-(E-hydroxyimitol)-5-nitro-6-methoxy-3-hexeneamide (NO-R). 6. SNAP-induced I(CCh) inhibition was effectively antagonized by Methylene Blue (1-100 nM), and mimicked by dibutyryl cGMP (db-cGMP) (0.5-1 mM), whereas two structurally distinct types of cGMP-dependent (G)-kinase inhibitor, N-(2-aminoethyl)-5-isoquinilinesulphonamide (H-8, 2.5 μM) and KT5823 (1 μM), failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor R(p)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS; 1 μM) itself caused a marked reduction in I(CCh). 7. SNAP (100 μM) or db-cGMP (100 μM) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8. These results suggest that inhibition of I(CCh) by NO donors involves a cGMP-dependent but G-kinase-independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.