alpha-chymotrypsin and linsidomine

1. We have investigated the correlation between relaxation and changes in cyclic nucleotide content of human tracheal smooth muscle (HTSM) in vitro following inhibitory non-adrenergic non-cholinergic (i-NANC) neural bronchodilator responses evoked by electrical field stimulation (EFS), and compared these with changes seen with sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and vasoactive intestinal peptide (VIP). The effects of N omega-nitro-L-arginine methyl ester (L-NAME), Methylene Blue and alpha-chymotrypsin (alpha-CT) were studied. 2. EFS (10 Hz, 1 ms, 40 V for 30 s) evoked a time-dependent relaxation accompanied by a concurrent rise in cGMP, both of which were maximal at 30 s and unaffected by epithelium removal. Levels of cAMP were more variable than those of cGMP and were not significantly changed at any time point. 3. SIN-1 (1 mM) and SNP (100 microM) also produced time-dependent relaxations which were maximal between 2 and 8 min, accompanied by concomitant rises in cGMP; however, these changes were larger than those associated with i-NANC relaxations. cAMP levels were unchanged at all time points. 4. EFS-evoked i-NANC relaxations and cGMP increases (time, t = 30 s) were inhibited by L-NAME. The effects were partially reversed by L-arginine (1 mM), but not by D-arginine. D-NAME and alpha-CT (2 u ml-1) had no effect on either relaxation or cGMP accumulation. Tetrodotoxin (TTX, 3 microM) inhibited both relaxation and cGMP accumulation. 5. VIP (1 microM) also produced a time-dependent relaxation associated with a concurrent rise in cAMP levels with no change in cGMP levels. 6. Methylene Blue (10 microM) partially inhibited EFS (10 Hz)-evoked i-NANC relaxation and cGMP accumulation, and almost completely inhibited both relaxation and cGMP accumulation evoked by SIN-1 (1 mM). Methylene Blue had no significant effect on relaxation or cGMP accumulation evoked by SNP (100 microM). 7. Neural i-NANC relaxations in HTSM are associated with a concurrent selective accumulation of cGMP which is unaffected by epithelium removal. This is inhibited in a stereoselective manner by L-NAME and mimicked by SNP and SIN-1; however, cGMP accumulation was greatly increased with SNP and SIN-1 suggesting compartmentalized changes in cGMP content. VIP also caused relaxation associated with an increase of cAMP; however, no evidence was found for VIP being involved in i-NANC relaxation. Hence nitric oxide (NO), or a NO-containing complex, appears to mediate i-NANC

Topics: Adenosine Monophosphate; Adolescent; Adult; Arginine; Bronchoconstriction; Child; Chymotrypsin; Cyclic GMP; Electric Conductivity; Epithelium; Female; Humans; Male; Methylene Blue; Middle Aged; Molsidomine; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Tetrodotoxin; Time Factors; Trachea; Vasoactive Intestinal Peptide; Vasodilator Agents

Human isolated central (5 to 12 mm) and peripheral (< 2 mm) bronchi were contracted with 3 microM histamine. Relaxations were then evoked by electrical field stimulation (EFS) (1 to 32 Hz, 1 ms, 12 V for 15 s in the presence of indomethacin, atropine, and propranolol). The magnitude, time-course, and frequency-response relationship of these nonadrenergic, noncholinergic (NANC) relaxations were similar in the central and the peripheral airways. NG-Nitro-L-arginine (L-NOARG) (10 microM) inhibited the tetrodotoxin-sensitive NANC relaxations in both central and peripheral bronchi, whereas the stereoisomer D-NOARG was without effect. This inhibition was reversed by L-arginine (1 mM) but not be D-arginine (1 mM). The nitric oxide donor compound, 3-morpholinosydnonimine (SIN-1), was equipotent at relaxing the central and peripheral airways. Vasoactive intestinal peptide (VIP), although it relaxed central airways, was virtually ineffective in relaxing the peripheral airways. In addition, the peptidase, alpha-chymotrypsin, at a concentration that blocked relaxations to VIP, was without effect on NANC relaxations in the central bronchi. The results support the following hypotheses: (1) both central and peripheral airways receive nonadrenergic relaxant innervation; (2) the relaxant response to electrical stimulation of this system is dependent on a pathway involving L-arginine; and (3) the relaxant response does not appear to involve VIP, but it may involve the production of nitric oxide.

Topics: Arginine; Bronchi; Chymotrypsin; Electric Stimulation; Humans; In Vitro Techniques; Molsidomine; Muscle Relaxation; Nitroarginine; Stereoisomerism; Vasoactive Intestinal Peptide; Vasodilator Agents