Evidence for the involvement of cgmp in neural bronchodilator responses in humal trachea

Transcription

1 3327 Journal of Physiology (1995), 483.2, pp Evidence for the involvement of cgmp in neural bronchodilator responses in humal trachea Jonathan K. Ward, Peter J. Barnes, Samad Tadjkarimi *, Magdi H. Yacoub* and Maria G. Belvisi Departments of Thoracic Medicine and * Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK 1. We have investigated the correlation between relaxation and changes in cyclic nucleotide content of human tracheal smooth muscle (HTSM) in vitro following inhibitory nonadrenergic 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 NW-nitro-L-arginine methyl ester (L-NAME), Methylene Blue and a-chymotrypsin (o-ct) were studied. 2. EFS (1 Hz, 1 ms, 4 V for 3 s) evoked a time-dependent relaxation accompanied by a concurrent rise in cgmp, both of which were maximal at 3 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 (1,uM) 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 = 3 s) were inhibited by L-NAME. The effects were partially reversed by L-arginine (1 mm), but not by D-arginine. D-NAME and a-ct (2 u ml-') had no effect on either relaxation or cgmp accumulation. Tetrodotoxin (ITX, 3/uM) inhibited both relaxation and cgmp accumulation. 5. VIP (1 ulm) also produced a time-dependent relaxation associated with a concurrent rise in camp levels with no change in cgmp levels. 6. Methylene Blue (1,UM) partially inhibited EFS (1 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 (1,UM). 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 responses in human trachea in vitro. Human tracheal smooth muscle (HTSM) possesses a neural response. This can be demonstrated in vitro by electrical bronchodilator response which is unaffected by either field stimulation (EFS) of isolated tissue preparations muscarinic or adrenergic receptor antagonists (Richardson (Richardson & Beland, 1976) and in vivo by reflex & Beland, 1976) and hence is termed the inhibitory non- stimulation of the airways (Lammers, Minette, McCusker, adrenergic non-cholinergic (i-nanc) bronchodilator Chung & Barnes, 1988). In HTSM the i-nanc response

2 526 J K. Ward and others J. Physiol appears to be solely mediated by nitric oxide (NO) (Belvisi, Stretton, Yacoub & Barnes 1992b; Belvisi et al. 1992a). However, there is speculation, as Bai & Bramley (1993) have suggested, that there is a significant Nw-nitro- L-arginine methyl ester (L-NAME)-resistant, and hence NO-independent, part of the i-nanc response in human bronchial smooth muscle. In guinea-pig airways the i-nanc response is mediated by both NO and vasoactive intestinal peptide (VIP; Tucker, Brave, Charalambous, Hobbs & Gibson, 199; Li & Rand, 1991; Ellis & Farmer, 1989a, b). However, in human airways there is no convincing evidence that VIP plays a role in i-nanc relaxation (Belvisi et at. 1992b; Bai & Bramley, 1993). In the absence of a selective VIP receptor antagonist that is effective in attenuating relaxation responses to VIP in the airways (Ellis & Farmer, 1989a), these experiments were carried out using the non-specific peptidase a-chymotrypsin (a-ct). a-ct abolishes relaxant responses to exogenously applied VIP in HTSM (Belvisi et al b), and inhibits the L-NAME-resistant i-nanc response in guinea-pig trachea (Ellis & Farmer, 1989b), but has no effect on i-nanc relaxations evoked by EFS in HTSM (Belvisi et at. 1992b). Paradoxically, immunohistochemical studies have shown human bronchial smooth muscle to have a dense VIPergic neural supply which decreases in density from proximal to distal bronchi (Laitinen, Partanen, Hervonen, Pelto-Huikko & Laitinen, 1985), and that both nitric oxide synthase (NOS) and VIP are present in intrinsic ganglia and nerves of HTSM, even being colocalized in a subpopulation of ganglia (Fischer, Hoffmann, Hauser-Kronberger, Mayer & Kummer, 1993). In vascular tissues both NO and nitrovasodilators cause relaxation of smooth muscle by activation of soluble guanylyl cyclase (Forstermann, Mulsch, Boheme & Busse, 1986), leading to a selective accumulation of cgmp (Rapoport & Murad, 1983), and furthermore, these increases and relaxations are inhibited by Methylene Blue (Martin, Villani, Jothianandan & Furchgott, 1985) and haemoglobin (Martin et at. 1985), and potentiated by cgmp-specific phosphodiesterase (PDE typev) inhibitors (Martin, Furchgott, Villani & Jothianandan, 1986). In some non-vascular smooth muscle the guanylyl-cyclasecgmp pathway has also been implicated as the mechanism of i-nanc relaxation. In the bovine retractor penis (BRP; Bowman & Drummond, 1984) and the mouse anococcygeus (Gibson & Mirzazadeh, 1989a, b) an endogenous nitrate has been implicated in i-nanc relaxations as the above drugs have been demonstrated to inhibit and/or potentiate these relaxations. In the rat, anococcygeus nitrovasodilators and NO-mediated i-nanc responses have also been shown to be associated with a concomitant selective rise in cgmp (Mirzazadeh, Hobbs, Tucker & Gibson, 1991), as in the BRP (Bowman & Drummond, 1984), and the opossum lower oesphageal sphincter (Barnette, Torphy, Grous, Fine & Ormsbee, 1989). In guinea-pig airways in vivo, indirect evidence has shown nitrovasodilators and i-nanc stimulation to cause relaxation which is partly via a cgmp-dependent mechanism (Ko & Lai, 1988). In contrast, VIP has been demonstrated to act via an adenylyl cyclase-camp pathway in a large number of tissues including guinea-pig trachea, where VIP-evoked relaxation of airway smooth muscle is associated with elevated camp levels (Fransden, Krishna & Said, 1978). We have studied the effects of i-nanc relaxant responses, elicited by EFS, on both cgmp and AMP levels in HTSM strips in vitro. To study the involvement of NO in these i-nanc relaxations we have compared these changes with those seen with the nitrosamine vasodilator sodium nitroprusside (SNP) and the sydnonimine 3-morpholinosydnonimine (SIN-1). SNP is converted to NO, either spontaneously (Waldman & Murad, 1987), or via a chemical reaction with cellular constituents containing sulfhydryl groups (Ignarro et al. 1981). SIN-1 also releases NO but is a prodrug which acts extracellularly by an oxidative, non-enzymatic process (Feelisch, Ostrowski & Noack, 1989). Both SNP and SIN-1 have been suggested to evoke relaxation via a cgmpguanylyl cyclase-dependent pathway in airway smooth muscle (Diamond, 1993) and this has been best characterized in canine airways (Zhou & Torphy, 1991; Jones, Lorenz, Warner, Katusic & Sieck, 1994), although in the case of SNP it appears a cgmp-independent mechanism of relaxation may also exist (Zhou & Torphy, 1991). We have also studied the effects of the NOS inhibitor N"-nitro-L-arginine (L-NAME; Rees, Palmer, Schulz, Hodson & Moncada, 199), the guanylyl cyclase inhibitor, Methylene Blue and the selective neuronal blocker tetrodotoxin (ITX) on the relaxations and changes in cyclic nucleotide content evoked by i-nanc responses, SNP and SIN-1. To study the involvement of VIP as a neurotransmitter involved in the i-nanc relaxation we have compared cyclic nucleotide changes seen during the i-nanc response with those seen with VIP added exogenously to the tissue. We have also studied the effects of the peptidase a-ct on the cyclic nucleotide changes seen during the i-nanc response. METHODS Donors Tissues were obtained from twenty-one normal non-smoking human donors for heart and/or heart-lung transplantation (age years, mean age 26f6 years, 12 male). These were termed normal tissues after macroscopic histological inspection and the previous medical histories showed there to be no evidence of chronic lung disease.

3 J. Physiol i-nanc responses increase cgmp in human airways 527 Tissue samples Ex vivo lung tissues were immediately immersed in oxygenated Krebs-Henseleit (KH) solution and cooled to 4 C for transport to the laboratory. The composition of KH was as follows (mm): NaCl, 118; KCI, 5 9; MgSO4, 1-2; CaCl2, 2-5; NaH2PO4, 1P2; NaHCO3, 25 5; and glucose, 5-6. Samples of smooth muscle were dissected from the trachea or main bronchus, with care taken to remove as much cartilage and connective tissue as possible. After preliminary experiments, the epithelium was removed by careful dissection and histology was used to confirm that this removal had not damaged the underlying submucosa. Smooth muscle was cut into strips (-2 mm wide) and weighed prior to measurement of isometric tension. Time taken for removal of the lungs from the donor to commencement of the experiment was generally 6-8 h. Measurement of contraction Tissue preparation. Smooth muscle strips were suspended by steel hooks in a 1 ml organ bath containing KH solution bubbled with 95 % 2-5% CO2 and maintained at 37 C (ph 7 4). Tissues were tied with silk thread to Grass FT-3 forcedisplacement transducers (Grass Instruments, MA, USA) for measurement of isometric changes in tension, recorded on a polygraph (Grass model 7D). Indomethacin (1 /um) and propranolol (1 /LM) were present throughout, to prevent any disruption of resting tension by endogenously produced prostaglandins or catecholamines, respectively. Tissues were left to equilibrate, with frequent washing, for 2 h and resting tension was adjusted to 2 g which was found to be optimal for measurement of changes in isometric tension. Electrital field stimulation. EFS was applied via two platinum wire field electrodes (-1 mm apart) placed on either side of the tissue. A Grass S 88 stimulator connected to a unity gain inverting amplifier provided biphasic square-wave impulses with a supramaximal voltage of 4 V at source and a pulse duration of 1 ms. To elicit i-nanc relaxations, EFS was applied at 1 Hz, a stimulation frequency which has been shown previously to evoke maximal L-NAME-sensitive neural i-nanc relaxations (Belvisi et al. 1992b). Protocol. EFS was applied to tissues in the presence of atropine (1 #M), to block cholinergic responses, and propranolol (1 1AM), to block any adrenergic responses, in order to elicit i-nanc relaxations. In some tissues the neural origin of these NANC relaxations was confirmed by blockade with tetrodotoxin (3 /tm). In preliminary experiments EFS was applied at 1 Hz (control EFS) and the maximum relaxation was noted. Tissues were then left for at least 3 min and EFS (1 Hz) was reapplied (test EFS); tissues were then removed at various time points (3, 1, 3, 6, 12, 24 and 6 s), and relaxation measured simultaneously. The tissues were blotted on filter paper to remove excess KH solution, snap frozen in liquid nitrogen and stored at -8 C for assessment of cyclic nucleotide content. In subsequent experiments the same protocol was used but tissues were cut down and tension measured at the 3 s time point, as this was shown to give the maximum increase in cgmp content. In other tissues the effects of L-NAME (1 AM) (+ L- and/or D-Arg (1 mm)), D-NAME (1 UM), TTX (3 /SM), a-ct (2 u ml-') and Methylene Blue (1 UM) on both relaxation and cgmp and/or AMP content were studied. Drugs were added after the control EFS (1 Hz) and at least 3 min before the test EFS (1 Hz). In a separate series of experiments the effects of SNP (1 /SM), SIN-1 (1 mm), and VIP (1 /SM) were studied both on relaxation and cyclic nucleotide content. Again tissues were cut down at various time points ( 5, 1, 2, 4, 8, 16 and 32 min) after drug addition and snap frozen in the same way as above. The effects of L-NAME (1 /um) and Methylene Blue (1O /LM), added 3 min before SNP and/or SIN-1, on relaxation, cgmp and AMP increases were also investigated. Measurement of cyclic nucleotide content Extraction of cyclic nucleotides. Frozen tissue samples were weighed and nucleotides were extracted by homogenization in 2 ml of ice-cold trichloroacetic acid (TCA; 5 M). Tubes were centrifuged (25 g for 15 min) at 4 C and 5,s1 of supernatant was removed. EDTA (5 u1l of 25 mm) was added to inhibit any Ca2+-Mg2+-ATPase activity present in the sample. TCA was extracted by vortex mixing of the samples with 5,1u of 1,1,2-trichlorotrifluoroethane: tri-n-octylamine (1:1, v/v), microfuging samples (15 g for 2 min) and removing 45 ul of the upper aqueous phase. Samples were then neutralized with 5 #zl of NaHCO3 (12 mm) and stored in a -2 C freezer for assessment of cyclic nucleotide content. Radioimmunoassay. Aliquots of the neutralized cyclic nucleotide-containing extract were diluted 5- to 5-fold in 1 mm sodium acetate buffer (ph 6 2) to a final sample volume of 5 lt1. Samples were acetylated by the consecutive addition of triethylamine (2 #1) and acetic anhydride (1 #1) and assessed for camp and/or cgmp content, in duplicate, using a radioimmunoassay (RIA) technique described by Brooker, Harper, Terasaki & Moylon (1979). In brief, 5 u1 of adenosine and/or guanosine-3',5'-cyclic monophosphate, 2-O-succinyl 3-['251]iodotyrosine methyl ester and 1 /il of anti-camp and/or cgmp antibody, both in 1 % bovine serum albumin (BSA), were added to 2 Al of the acetylated sample. Samples were then vortex mixed and left at 4 C overnight to allow equilibration. Free and antibody-bound [1251]cGMP and/or camp were then separated using charcoal precipitation in 1 mm phosphate buffer. Samples were centrifuged (25 g for 3 min) and 7 #1 of the supernatant was removed for assessment of antibody-bound I1251]cAMP and/or cgmp by y-counting (5 min, Autogamma 5, Packard Instrument Co. Inc., Downers Grove, IL, USA). The cgmp and/or camp content of the original samples was then assessed by comparison of the counts from the unknown samples with those from a set of standard samples containing known amounts of cgmp (-4 fmol sample-') or camp (-16 fmol sample-'). For these assays the detection limits (12) for egmp and camp were approximately 2 and 4 fmol, respectively, and the sensitivity (IC5) approximately 15 and 35 fmol, respectively. Reagents and drugs The following drugs and reagents were used: indomethacin, tetrodotoxin (TTX), Methylene Blue, L-arginine, D-arginine, papaverine, a-chymotrypsin (a-ct), bovine serum albumin (BSA; fraction V), cgmp (sodium salt), camp (sodium salt), EDTA, sodium nitroprusside, anti-camp antibody, anti-cgmp antibody (Sigma Chemical Co., Poole, Dorset, UK); Nw-nitro-Larginine methyl ester and NW-nitro-D-arginine methyl ester (L- and D-NAME; Bachem Feinchemikalien AG, Bupendorf, Switzerland); atropine sulphate (Pharma Hameln GMBH,

4 528 J K. War-d cand others J. Physiol Germany); propranolol hydrochloride (ICI plc, Macclesfield, Cheshire, UK); guanosine 3',5'-cyclic phosphoric acid, 2'- -succinyl 3-['251]iodotyrosine methyl ester and adenosine 3',5'-cyclic phosphoric acid, 2'-O-succinyl 3-['25I]iodotyrosine methyl ester, both with activity -74 TBq mmol-f (Amersham International plc, Amersham, UK); SIN-i was a generous gift from Dr Kunstmann (Cassella AG, Frankfurt, Germany). KH solution was freshly made each day. Aliquots of drugs were dissolved in distilled water and stored at -2 C for no longer than 1 week. Indomethacin was made up in an alkaline phosphate buffer (ph 7 8) of the following composition (mm): KH2PO4, 2; Na2HPO4, 12. Drug additions did not exceed I % of the bath volume. All concentrations refer to final bath concentrations. Statistical analysis Data are expressed as means + S.E.M. Relaxation responses are expressed as milligrams of tension in most experiments, or, as a percentage of the preceding control 1 Hz EFS in the case of experiments looking at the effects of drugs on cyclic nucleotide accumulation and relaxation evoked by i-nanc relaxations. Due to the low sample numbers used in these studies (generally n = 5-6), it is not possible to determine whether the data are normally distributed, hence a more conservative non-parametric Mann-Whitney U test was used (Number Cruncher Statistical System, UT, USA). This was used to compare baseline levels of cyclic nucleotides with levels seen with EFS or in the presence of SNP, SIN-i and VIP. It was also used to compare relaxations and cyclic nucleotide accumulation in groups of tissues treated with L-NAME (± L- and/or D-Arg), D-NAME, a-ct, TTX or Methylene Blue with those seen in control groups without drug additions. Statistical analysis was not performed on relaxations seen with EFS, SNP, SIN-i and VIP as it was inappropriate to compare these relaxations with the zero baseline preceding EFS and/or drug addition. P values < 5 were considered significant: * P< 5, ** P< 1, *** P< O1; n.s. represents statistically not significant values. n values represent the number of separate donors studied. A cm E c ) B C 4 _ c a) C) _. a- E CI)) c ) C) -E 5 _5 E [ <- -I EFS Time (s) Figure 1. Time course for i-nanc relaxation and cyclic nucleotide accumulation following EFS in HTSM Time course for i-nanc relaxation and cyclic nucleotide accumulation following EFS (1 Hz, ms, 4 V applied between t = and 3 s) in HTSM. Time course of i-nanc relaxation evoked by EFS (A), time course of cgmp accumulation (B), time course of camp accumulation (C). Data represent means + S.E.M. for at least n = 5 donors. * P < 5, ** P < 1, ***P < 1 vs. t =.

5 J. Physiol i-nanc responses increase cgmp in human airways 529 RESULTS Time course of i-nanc relaxation and cyclic nucleotide accumulation following EFS EFS (4 V, 1 ms, 1 Hz for 3 s) in the presence of atropine and propranolol (both 1 /SM) produced a transient time-dependent relaxation (Fig. 1A) which was maximal at 3 s ( mg, n = 5) and returned to baseline between 4 and 1 min. This relaxation was accompanied by a concurrent elevation of cgmp levels (Fig. 1B) which was also maximal at 3 s (t = : cgmp = fmol (mg tissue)-', n = 1; t = 3 s: cgmp = fmol (mg tissue)-', n = 1; P< 1 vs. t = ) and had returned to baseline between 2 and 4 min (t=4 min: cgmp = fmol (mg tissue)-', n = 5; n.s. vs. t = ). Levels of camp were generally more variable than those of cgmp (t= : camp = fmol (mg tissue)-', n = 1) and there was no significant change in levels throughout the i-nanc relaxation (Fig. 1C). As the 3 s time point gave maximal i-nanc relaxations and cgmp accumulation this was chosen as the time point for further investigation. The effect of epithelium removal on relaxation and cyclic nucleotide accumulation following EFS We compared i-nanc relaxation and cyclic nucleotide accumulation, evoked by EFS (4 V, 1 ms, 1 Hz for 3 s) in tissues which were either epithelium denuded or intact. At the 3 s time point, relaxation was unaffected by epithelium removal (1 Hz EFS: + epithelium, mg, n = 4; - epithelium, mg, n = 6; n.s. compared with + epithelium). Neither basal nor stimulated levels of cgmp (t = : + epithelium, P fmol (mg tissue)-', n = 4; - epithelium, fmol (mg tissue)-', A _C.o 8 N 1 F 8 k 6 V T1 l17 T7- T1 -r 4 F 2 - B 3 ** * 25 _ r (D C t* 2 E TL I-_ IL *** L.^ (9*E iet Control t=o s ** * r1 Control + L-NAME + L-NAME + L-NAME + D-NAME t=3 s + L-Arg + D-Arg + a-ct +TTX EFS Figure 2. Effects of L-NAME, TTX and a-ct on i-nanc relaxation in HTSM The effect of L-NAME (± L-/D-Arg), D-NAME, TTX and a-ct on i-nanc relaxation (A), and cgmp accumulation (B) following EFS (1O Hz, 1 ms, 4 V for 3 s) in HTSM. Data represent the means + S.E.M. of at least n = 5 donors. * P< 5, ** P< '1, *** P< 1 vs. relaxation and/or cgmp accumulation at t = 3 s time point.

6 53 J K. Ward and others J. Physiol n = 6; n.s. vs. + epithelium. t = 3 s: + epithelium, fmol (mg tissue)-', n = 4; - epithelium, P8 fmol (mg tissue)-', n = 6; n.s. vs. + epithelium) or camp (data not shown) were affected by removal of the epithelium; however, in subsequent experiments tissues were used without epithelium. The effects of L-NAME, TTX and oc-ct on relaxation and cyclic nucleotide accumulation following EFS The effects of L-NAME, TTX and a-ct on the relaxation and cyclic nucleotide accumulation following i-nanc responses evoked by EFS (4 V, 1 ms, 1 Hz for 3s; Fig. 2) were studied. Again the effects of these drugs were studied at the 3s time point as this was shown to produce maximal relaxation and cgmp accumulation. EFS-evoked relaxation (1P12 + 8&% of control, n = 6) and cgmp accumulation ( P7 fmol (mg tissue)') were both attenuated by L-NAME (1,uM) (relaxation, % of control; cgmp, fmol (mg tissue)-'; both n = 5; both P < 1 vs. control) but not by D-NAME (1,lM). This inhibition was partially reversed in a stereoselective manner by L-arginine (1 mm) (relaxation: % of control, n = 5, P < 1 vs. + L-NAME; cgmp: fmol (mg tissue)-', n = 5, P< 5 vs. + L-NAME) but not by D-arginine (1 mm). a-ct (2 u ml-') had no significant effect on either relaxation or cgmp accumulation. TTX (3/uM) significantly inhibited both relaxation ( '9% of control, n = 5, P < 1 vs. control) and cgmp accumulation ( P1 fmol (mg tissue)-', n = 5, P < 1 vs. control). camp levels were again unchanged in all of the groups tested (data not shown). Time course of relaxation and cyclic nucleotide accumulation following addition of SIN- I or SNP Maximally effective concentrations of SIN-1 (1 mm) or SNP (1/,M) produced time-dependent relaxations of A B 2 ^ 4 E 6 a 8 X 1 a) r 12 14Q Ca)( O 12 8 ; 1. E 8 6 E C CD a): 3 25 a. E 2 E 15 1 i-iii I E SIN (a co a: c F 6 c 1= i 5 o4.- 3 i E 2 Figure 3. Time course for relaxation and cyclic nucleotide accumulation following a single addition of SIN- I Time course for relaxation and cyclic nucleotide accumulation following a single addition (at t = ) of 3-morpholinosydnonimine (SIN-1, 1 mm) in HTSM. Time course of relaxation (A), time course of cgmp accumulation (B) and time course of camp accumulation (C). Insets show cgmp accumulation accompanying relaxation at the lowest time points (< 1 min). Data represent means + S.E.M. for at least n = 5 donors. * P< 5, ** P< 1, *** P< 1 vs. t =. 1 I I

7 J. Physiol i-nanc responses increase cgmp in human airways 531 HTSM (Figs 3A and 4A). Maximum relaxations were approximately equal in magnitude and were both maximal between 2 and 8 min (8 min, SIN-1: mg, n = 5; SNP, mg, n = 5). They were accompanied by concurrent rises in cgmp (SIN-1: t =, 13X fmol (mg tissue)-1, n = 5; t= 8 min, fmol (mg tissue)-', n = 5; P < -1 compared with t=. SNP: t=, fmol (mg tissue)-', n = 5; t = 8 min, 264X4 + 48X4 fmol (mg tissue)-', n = 5; P < 1 compared with t = ) (Figs 3B and 4B). camp levels were unchanged for all time points studied (Figs 3C and 4C). The effect of Methylene Blue on relaxation and cgmp accumulation following EFS-induced i-nanc responses, SIN-I and SNP The effect of Methylene Blue (1 UM) was studied on the relaxation and cyclic nucleotide accumulation following i-nanc responses evoked by EFS (4 V, 1 ms, 1 Hz for 3 s) and relaxations elicited by SIN-1 (1 mm) or SNP (1 um) (Fig. 5) in HTSM. Methylene Blue produced partial inhibition of both i-nanc relaxation ( % of control, n = 5, P < 1 vs. control) and cgmp accumulation ( fmol (mg tissue)-' tissue, n = 5, P < 5 vs. control) evoked by EFS at the 3 s time point. Furthermore, Methylene Blue produced complete inhibition of both relaxation ( mg, n = 5, P < 1 vs. SIN-1 alone) and cgmp accumulation ( fmol (mg tissue)-', n = 5, P < 1 vs. SIN-1 alone) in response to SIN-I (1 mm, added at t = ) at the 2 min time point (Fig. 5). In contrast, Methylene Blue (1 /SM) had no significant effect on either relaxation ( mg, n = 5, n.s. vs. SNP alone) and cgmp accumulation ( fmol (mg tissue)-1, n = 5, n.s. vs. SNP alone) in response to SNP (1 ftm, added at t= ) at the 2 min time point (Fig. 5). A B 2 a 4 E 6 O 8 x 1 G) ^ 3 CQ ) 25 -co,,-r- 2 L E 15 (D C 4- a a1) D3 CU) a- E <- E IQ...T **I/ ** SNP o E 2 - c = 4 - x _ C U) E _4 c NE 2 - o 1 II 1 Figure 4. Time course for relaxation and cyclic nucleotide accumulation following a single addition of sodium nitroprusside Time course for relaxation and cyclic nucleotide accumulation following a single addition (at t = ) of sodium nitroprusside (SNP, 1,UM) in HTSM. Time course of relaxation (A), time course of cgmp accumulation (B) and time course of camp accumulation (C). Insets show cgmp accumulation accompanying relaxation at the lowest time points (< I min). Data represent means + S.E.M. of at least n=5donors.*p<o5,**p<oot,***p<oo1 vs. t=.

8 532 J K. Ward and others J. Phy-siol The effect of L-NAME on relaxation and cgmp accumulation following addition of SIN-I or SNP The effect of L-NAME (1 /SM) on the relaxation and cyclic nucleotide accumulation evoked by SIN-1 (1 mm) or SNP (1 /SM) (Fig. 5) in HTSM was studied. L-NAME had no significant effect on either relaxation (SIN-1, mg; SNP, mg; both n = 5; both n.s. vs. control) or cgmp accumulation (SIN-1, fmol (mg tissue)-; SNP, fmol (mg tissue)-1; both n = 5; both n.s. vs. control) (Fig. 5) at the 2 min time point. Time course of relaxation and cyclic nucleotide accumulation evoked by VIP The effect of VIP (1 /SM) was studied on both relaxation and cyclic nucleotide accumulation in HTSM. VIP (1 usm) produced a time-dependent relaxation (Fig. 6) (t = 8 min: mg, n = 6) which was slower in onset than i-nanc or SNP- and/or SIN-1-induced relaxations and associated with a concurrent rise in camp levels (t = : fmol (mg tissue)-', n = 6; t = 8 min: fmol (mg tissue)-', n = 6; P < 1 vs. t = ; Fig. 6), with no change in cgmp levels (data not shown). DISCUSSION We have shown that basal levels of camp and cgmp present in HTSM are detectable by a simple acetylation radioimmunoassay. Basal levels of cgmp (-1 fmol (mg tissue)') are comparable with those found in human bronchi ('-1 fmol (mg tissue)-' tissue; Gaston et al. 1994). Basal levels of camp were 15- to 2-fold higher than those of cgmp (-15-2 fmol (mg tissue)-'); however, there is no direct comparison with other studies in human airways. We studied the changes in camp and cgmp content of HTSM during EFS-evoked i-nanc relaxations in vitro, to elucidate the contribution of NO and VIP in this response. Studies in vascular (Knowles & Moncada, 1992; Ignarro, 1992) and non-vascular (Rand, 1992) smooth muscle have suggested that NO produces relaxation via A E x -5 CD o B I} C~U) ud u c} 1 (5- g E 5 Control + SNP + SIN-1 (t = ) (t=2 min) (t=2 min) Figure 5. The effect of Methylene Blue and L-NAME on relaxation and cgmp accumulation following SIN- I or SNP The effect of Methylene Blue (1,m, cross-hatched columns) and L-NAME (1,m; checked columns) on relaxation (A) and cgmp accumulation (B), following SIN-1 (1 mm) or SNP (1 /M) in HTSM. Data represent means + S.E.M. of at least n = 5 donors. * P < 5, ** P < 1 vs. cgmp content at t =. tt P < 1 vs. relaxation and/or cgmp accumulation at t = 2 min time point.

9 J. Physiol i-nanc responses increase cgmp in human airways 533 activation of soluble guanylyl cyclase and selective accumulation of cgmp. Hence if NO is acting as the i-nanc transmitter in HTSM then, by inference, a similar mechanism may be involved. If cgmp is the intracellular second messenger then increases in cgmp should precede or be coincident with relaxation elicited by EFS (Sutherland, Robinson & Butcher, 1968). EFS in the presence of atropine and propranolol produced a transient time-dependent i-nanc relaxation which was associated with a concurrent increase in cgmp content with cgmp increasing 2*5-fold at the maximum relaxation. There was no significant change in camp content. There have been no previous reports of changes in cyclic nucleotide levels associated with i-nanc relaxations in airway smooth muscle, hence comparisons of increases in airway tissues is not possible. In other nonvascular smooth muscle preparations, where cyclic nucleotide changes associated with i-nanc relaxations have been investigated, increases in cgmp are variable. In the rat, anococcygeus i-nanc stimulation produced no change in cgmp unless a permissive dose of SNP or a phosphodiesterase (PDE) type V inhibitor was present (Mirzazadeh et al. 1991). In the opossum lower oesphageal sphincter (Barnette et al. 1989) and the BRP (Bowman & Drummond, 1984) i-nanc relaxations were accompanied by 3- and 2-fold increases in cgmp levels, respectively. Both basal and stimulated levels of cgmp associated with i-nanc relaxation in HTSM are unaffected by epithelium removal, suggesting that changes are localized to smooth muscle and are not modulated by epitheliumderived factors. Hence it appears that our selective increases in cgmp are in line with increases in other nonvascular smooth muscle where NO has been postulated to mediate i-nanc relaxation. Further evidence that NO is the mediator of the i-nanc response in HTSM was obtained by use of the NOS inhibitor L-NAME (Rees et al. 199). L-NAME produced complete inhibition of both i-nanc relaxation and cgmp, consistent with an NO-mediated response. Inhibition was partially reversed by an excess of the NOS substrate L-Arg but not by its inactive enantiomer D-Arg. D-NAME, the inactive optical isomer of L-NAME (Rees et al. 199), had no effect on either relaxation or cgmp accumulation. There are several possible reasons why L-Arg evokes only partial reversal. L-NAME may be strongly bound to the NOS enzyme and hence a higher concentration (> 1 mm) of L-Arg is required to reverse its effects, or it could be that L-NAME and L-Arg have different abilities to access intact cells. However, this partial reversal of L-NAME-evoked inhibition by L-Arg is consistent with other studies of the i-nanc response in HTSM (Belvisi et al. 1992b; Ward et al. 1993). Evidence has recently arisen that L-NAME, as well as inhibiting NOS, can act as a muscarinic (Ml, M2 and M3) antagonist (Buxton, Cheek, Eckman, Westfall, Sanders & Keef, 1993); however, this does not occur in all tissue types A 2 - cm 4 Figure 6. Time course for relaxation and camp accumulation following a single addition of vasoactive intestinal peptide Time course for relaxation and camp accumulation following a single addition (at t = ) of vasoactive intestinal peptide (VIP, 1,UM) in HTSM. Time course of relaxation (A), time course of camp accumulation (B). Data represent means + S.E.M. for n = 6 donors. * P< 5, ** P< 1 vs. t =. B * 6 a) 8 _4- a1) D3 4- (n) CO)z -E i' E I I- 3 F 2 I 1 I ± 1 o

10 534 J K. Ward and others J. Physiol (Sideso, Tucker & Gibson, 1994). In HTSM, L-NAME appears not to act as a muscarinic antagonist, as it has no effect on contractile responses to exogenous acetylcholine (suggesting no effect on M3 receptors), and no effect on [3H]acetylcholine release (suggesting no effect on M2 receptors) (Ward et al. 1993). Furthermore, the effects of L-NAME are reversed by L-Arg and therefore we have assumed that L-NAME is acting as a NOS inhibitor in HTSM. To confirm that exogenous NO evokes relaxation of HTSM by elevation of cgmp levels we studied the effects of the nitrovasodilator SNP and the NO donor SIN-1, both of which release NO, both on relaxation and cyclic nucleotide accumulation. SIN-1 and SNP, at maximally effective concentrations (Ward et al. 1994), produced time-dependent relaxations accompanied by a concomitant selective increase of cgmp levels with no change in camp levels. These selective increases in cgmp levels are consistent with those of canine airways where both SNP (Zhou & Torphy, 1991) and SIN-1 (Jones et al. 1994) evoke selective accumulation of cgmp with no increase in camp levels. SNP produced a 2-fold increase, comparable to that seen in canine airways (-18-fold; Zhou & Torphy, 1991). SIN-1 (1 mm) produced a 1-fold increase, somewhat larger than that seen in canine trachea (-2-fold); however, in canine airways a lower concentration (1 /M) was studied (Jones et al. 1994). Although both SNP and SIN-1 produced a selective rise of cgmp, suggesting a similar mechanism of relaxation to that of i-nanc responses, increases are exaggerated. These exaggerated increases were not without precedent as Bowman & Drummond (1984) demonstrated, that in the BRP, i-nanc-evoked cgmp increases (- 2-fold) were smaller than those with SNP (- 4- to 5-fold). This suggests that there may be compartmentalized guanylyl cylcase and/or cgmp pools intracellularly in HTSM, which are affected differentially by the i-nanc transmitter and nitrovasodilators. Another explanation is that SNP and SIN-1 are stimulating cgmp production in other non- HTSM cell types (e.g. fibroblasts, vascular smooth muscle cells) present in our preparation. The cgmp increases and relaxations evident with SIN-1 and SNP were unaffected by L-NAME demonstrating that these drugs are having their effects directly and not via activation of NOS. VIP also produced a time-dependent relaxation, but of slower onset to that evoked by EFS. VIP-induced relaxations were associated with a concomitant increase of camp levels with no change in cgmp levels, as in guineapig trachea (Fransden et al. 1978). camp levels increased (2-fold at the latest time point studied and this increase was comparable to that seen in guinea-pig trachea (--17-fold increase; Fransden et al. 1978). We have found no evidence for a role of camp in neurally mediated i-nanc responses, suggesting VIP is not involved in this response. However, camp levels are more variable than cgmp levels, measured in the same tissue extracts. Therefore, it is possible that if VIP was released as an i-nanc transmitter, then small changes in camp content may not be detected as the signal: noise ratio would be too low. To elucidate further the role of VIP in the i-nanc response, the effects of a-ct, which abolishes relaxations to exogenous VIP in HTSM (Belvisi et al. 1992b) and inhibits the VIP-mediated i-nanc neural response in guinea-pig trachea (Ellis & Farmer, 1989b), was studied. a-ct had no effect on either relaxation or cgmp accumulation evoked by EFS suggesting that VIP does not cause relaxation directly or via release of a second transmitter which could act via the cgmp pathway. This appears to be contradictory as human airways possess a rich VIPergic neural supply (Laitinen et at. 1985) and we have demonstrated that VIP can relax our preparations. It is possible that VIP present in airway nerves may be rapidly degraded once released and so inactivated before causing smooth muscle relaxation. Evidence suggests that human airway relaxations to exogenous VIP in vitro are potentiated by peptidase inhibitors (e.g. phosphoramidon, captopril and bestatin), suggesting that endogenous peptidase activity is high (Tam, Franconi, Nadel & Caughey, 199). Alternatively, it is possible that the VIPergic neural supply may be associated with blood vessels and not HTSM, as VIP is a potent vasodilator in human pulmonary vessels (Greenberg, Rhoden & Barnes, 1987). We have shown that relaxation of HTSM can occur by either camp- or cgmp-dependent mechanisms and that different stimuli evoke relaxation by a discrete second messenger system. This suggests that the cyclic nucleotide changes seen are causal in relaxation and not as a consequence. To study further the causal nature of cgmp accumulation in relaxation we studied the effect of Methylene Blue on relaxation and cgmp accumulation evoked by i-nanc responses, SIN-1 and SNP. Methylene Blue has been postulated to be an inhibitor of soluble guanylyl cyclase activity (Tremblay, Gerzer & Hamet, 1988), and inhibits cgmp accumulation and relaxation evoked by nitrovasodilators and NO donors in both vascular (Martin et al. 1985) and non-vascular smooth muscle, including canine trachea (Jones et al. 1994). In our experiments Methylene Blue significantly inhibited cgmp accumulation and relaxation evoked by SIN-1, partially inhibited cgmp accumulation and relaxation evoked by EFS and, although it slightly reduced cgmp accumulation evoked by SNP, had no effect on relaxation. These results are difficult to interpret, but are not unprecedented. Gaston et al. (1994) showed that in human bronchial smooth muscle cgmp increases, evoked by S-nitrosothiols are inhibited by Methylene Blue, whereas relaxations were not, and in porcine bronchi Methylene Blue was found to have no effect on relaxations evoked by SIN-1 or SNP (Bynoe,

11 J. Physiol i-nanc responses increase cgmp in human airways Stuart-Smith & Hirshman, 1992). Zhou & Torphy (1991) demonstrated that Methylene Blue inhibited relaxation and cgmp accumulation evoked by the nitrovasodilators S-nitroso-N-acetyl-penicillamine and glyceryl trinitrate, and inhibited SNP-induced cgmp accumulation, but paradoxically, potentiated SNP-induced relaxation in canine trachealis. In the same tissue Jones et al. (1994) demonstrated that Methylene Blue could inhibit both cgmp accumulation and relaxation evoked by SIN-1. Hence, although these NO-releasing compounds evoke cgmp accumulation, it may be that this is not a causative factor for relaxation or is only partially responsible for relaxation. Alternatively, Methylene Blue may not be acting as a soluble guanylyl cyclase inhibitor, but may instead have non-specific effects on smooth muscle. Finally, it is possible that Methylene Blue may actually mop up extracellularly released NO and if this was the case this could explain our results. SIN-1, which releases its NO extracellularly (Feelisch et al. 1989), would be most sensitive to Methylene Blue, whereas neurally released NO, with smaller distances to diffuse across the synaptic cleft, would be less sensitive. SNP can release NO spontaneously in a similar manner to SIN-1, but can also react with membrane-bound sulfhydryl groups to liberate NO (Ignarro et al. 1981), although it is not clear if this is intra- or extracellularly. Either way, NO would be formed either in, or in close apposition to, the airway smooth muscle, and so would be the least sensitive to Methylene Blue. This mechanism is purely speculative at present, and future studies using haemoglobin, which is thought to act by scavenging NO extracellularly, may provide further explanations for the differential effects of Methylene Blue on SNP, SIN-1 and i-nanc relaxations. We have demonstrated that, in HTSM in vitro, neurally mediated i-nanc responses are associated with a concomitant selective rise of cgmp, apparently associated with the smooth muscle itself. Relaxation and cgmp accumulation are inhibited in a stereoselective manner by NOS inhibitors, suggesting that NO or a NO-containing compound (e.g. S-nitrosothiol) mediates this response. Relaxations evoked by SIN-1 and SNP are also associated with cgmp increases, but of greater magnitude than those seen with i-nanc relaxations. VIP also evokes relaxation, but this is associated with selective camp accumulation. We found no evidence for a VIP-mediated i-nanc response. These data provide compelling evidence for a causal role of NO-induced cgmp accumulation in i-nanc relaxation, and cgmp accumulation in relaxations evoked by both nitrosamines and sydnonimines. However, the differential effects of Methylene Blue cast some doubts on this proposal. To characterize further the causal role of cgmp accumulation in relaxation it will be necessary to study the effects of PDE V inhibitors (e.g. zaprinast), other guanylyl cyclase inhibitors and analogues of cgmp, both on intact HTSM and cell-free guanylyl cyclase extracts. BAI, T. R. & BRAMLEY, A. M. (1993). Effect of an inhibitor of nitric oxide synthase on neural relaxation of human bronchi. Journal of Applied Physiology 264, L BARNETTE, M., TORPHY, T. J., GROUS, M., FINE, C. & ORMSBEE, H. S. (1989). Cyclic GMP: a potential mediator of neurally- and drug-induced relaxation of opossum lower oesphageal sphincter. Journal of Pharmacology and Experimental Therapeutics 249, BELVISI, M. G., STRETTON, C. 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Journal of Pharmacology and Experimental Therapeutics 258, Acknowledgements We would like to thank Dr Mark Giembycz for his help in assessment of cyclic nucleotide content. Funded by the National Asthma Campaign and by a Wellcome Trust Fellowship for M.G.B. Received 27 April 1994; accepted 4 November 1994.