Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl- currents by NO donors in cat tracheal smooth muscle.
1. The effects of NO donors on Ca2+-dependent Cl- currents (ICl(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 microM) induced a transient inward current (ICCh) with a reversal potential of about -20 mV. Activation of ICCh 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-(diethylamino)methyl)-1-piperidinylacetyl)-5, 11-dihydro-6H-pyrido (2,3beta) (1,4)benzodiazepine-6-one (AF-DX 116) or pirenzepine at concentrations of up to 1 microM were almost ineffective. 3. Chloride channel/transporter blockers such as DIDS (100 microM), anthracene-9-carboxylic acid (9-AC, 100 microM) and niflumic acid (100 microM) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 microM), Zn2+ (500 microM) or Gd3+ (500 microM), were without effect. 4. Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 microM) 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 ICCh. These results suggest that ICCh is ICl(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 ICCh dose dependently (IC50, approximately 10 microM). 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 ICCh 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 microM) and KT5823 (1 microM), failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor Rp-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS; 1 microM) itself caused a marked reduction in ICCh. 7. SNAP (100 microM) or db-cGMP (100 microM) 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 ICCh 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.
Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan.
Y
Y
Waniishi
R
R
Inoue
H
H
Morita
N
N
Teramoto
K
K
Abe
Y
Y
Ito
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002415:Cats; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008751:Methylene Blue / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000502:physiology; D012433:Ryanodine / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014132:Trachea / Q000166:cytology; D015032:Zinc / Q000494:pharmacology
C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D000873:Anthracenes; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; D020030:Nitric Oxide Donors; D010726:Phosphodiesterase Inhibitors; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C110881:S-nitro-N-acetylpenicillamine; D013873:Thionucleotides; D012433:Ryanodine; D003995:Dibutyryl Cyclic GMP; D002110:Caffeine; C010030:9-anthroic acid; D002217:Carbachol; D019204:GTP-Binding Proteins; D010396:Penicillamine; D006152:Cyclic GMP; D015032:Zinc; D002118:Calcium; D008751:Methylene Blue