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PubMed1997

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery.

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 83.6 +/- 3.6%; n = 8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC50 = 6.31 +/- 0.06, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 microM) greatly reduced the Rmax values for EDHF-mediated relaxations to carbachol (control, 93.2 +/- 0.4%; SR 141716A, 10.7 +/- 2.5%; n = 5; P < 0.001) and A23187 (control, 84.8 +/- 2.1%; SR 141716A, 3.5 +/- 2.3%; n = 6; P < 0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting Rmax. 3. Precontraction with 60 mM KCl significantly reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 68.8 +/- 5.6% versus 17.8 +/- 7.1%), A23187 (control 71.4 +/- 6.1% versus 3.9 +/- 0.45%) and anandamide (control 71.1 +/- 7.0% versus 5.2 +/- 3.6%). Similar effects were seen in the presence of 25 mM K+. Incubation of vessels with pertussis toxin (PTX; 400 ng ml-1, 2 h) also reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 63.5 +/- 7.5% versus 9.0 +/- 3.2%), A23187 (control 77.0 +/- 5.8% versus 16.2 +/- 7.1%) and anandamide (control 89.8 +/- 2.2% versus 17.6 +/- 8.7%). 4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 microM) significantly potentiated (P < 0.01), to a similar extent (approximately 2 fold), relaxation to A23187 (pEC50: control, 6.45 +/- 0.04; PMSF, 6.74 +/- 0.10; n = 4) and anandamide (pEC50: control, 6.31 +/- 0.02; PMSF, 6.61 +/- 0.08; n = 8). PMSF also potentiated carbachol responses both in the presence (pEC50: control, 6.25 +/- 0.01; PMSF, 7.00 +/- 0.01; n = 4; P < 0.01) and absence (pEC50: control, 6.41 +/- 0.04; PMSF, 6.88 +/- 0.04; n = 4; P < 0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC50: control, 7.51 +/- 0.06; PMSF, 8.00 +/- 0.05, n = 4, P < 0.001). 5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K+ channel blocker tetraethylammonium (TEA; 1 mM) (pEC50: control, 6.19 +/- 0.01; TEA, 5.61 +/- 0.01; n = 6; P < 0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to A23187 (pEC50: control, 6.47 +/- 0.04; TEA, 6.41 +/- 0.02, n = 4) or on anandamide (pEC50: control, 6.28 +/- 0.06; TEA, 6.09 +/- 0.02; n = 5). TEA (10 mM) significantly (P < 0.01) reduced the Rmax for anandamide (control, 94.3 +/- 4.0%; 10 mM TEA, 60.7 +/- 4.4%; n = 5) but had no effect on the Rmax to carbachol or A23187. 6. BaCl2 (100 microM), considered to be selective for blockade of inward rectifier K+ channels, had no significant effect on relaxations to carbachol or A23187, but caused a small shift in the anandamide concentration-response curve (pEC50: control, 6.39 +/- 0.01; Ba2+, 6.20 +/- 0.01; n = 4; P < 0.01). BaCl2 (1 mM; which causes non-selective block of K+ channels) significantly (P < 0.01) attenuated relaxations to all three agents (pEC50 values: carbachol, 5.65 +/- 0.02; A23187, 5.84 +/- 0.04; anandamide, 5.95 +/- 0.02; n = 4 for each). 7. Apamin (1mu M), a selective blocker of small conductance, Ca2+-activated, K+ channels (SKCa), 4-aminopyridine (1mM), a blocker of delayed rectifier, voltage-dependent, K+ channels (Kv), and ciclazindol (10mu M), an inhibitor of Kv and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8. Glibenclamide (10mu M), a KATP inhibitor and charybdotoxin (100 or 300nM), a blocker of several K+ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50nM), an inhibitor of large conductance, Ca2+-activated, K+ channels (BKCa), had no significant effect on the relaxation responses, either alone or in combination with apamin (1muM). Also, a combination of apamin (1muM) with either glibenclamide (10muM) or 4-aminopyridine (1mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to A23187 or anandamide. 9. A combination of apamin (1muM) with charybdotoxin (100nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1muM) and charybdotoxin (300nM) together consistently inhibited the response to A23187, while apamin (1muM) and ciclazindol (10muM) together inhibited relaxations to both carbachol and A23187. None of these toxin combinations had any significant effect on relaxation to anandamide. 10. It was concluded that the differential sensitivity to K+ channel blockers of EDHF-mediated responses to carbachol and A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K+ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K+ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.

4-Aminopyridine↗
PubMed1998

Mechanisms of relaxations of bovine isolated bronchioles by the nitric oxide donor, GEA 3175.

1. The present study was designed to investigate the effects and mechanisms of relaxation induced by the nitric oxide (NO) donor, GEA 3175 (a 3-aryl-substituted oxatriazole derivative) on bovine bronchioles (effective lumen diameter 200-800 microm) suspended in microvascular myographs for isometric tension recording. 2. In segments of bovine bronchioles contracted to 5-hydroxytryptamine, GEA 3175 (10(-8)-10(-4) M) induced concentration-dependent reproducible relaxations. These relaxations were slow in onset compared to other NO-donors such as 3-morpholinosydonimine-hydrochloride (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 3. In 5-hydroxytryptamine-contracted preparations the order of relaxant potency (pD2) was: salbutamol (7.80) > GEA 3175 (6.18) > SIN-1 (4.90) > SNAP (3.55). In segments contracted to acetylcholine, the relaxant responses were reduced and GEA 3175 relaxed the bronchioles with pD2 = 4.41 +/- 0.12 and relaxations of 66 +/- 10% (n = 4), while SNAP and salbutamol caused relaxations of 19 +/- 6% (n = 4) and 27 +/- 6% (n = 8) at the highest concentration used, respectively. 4. Oxyhaemoglobin (10(-5) M), the scavenger of nitric oxide, caused rightward shifts of the concentration-relaxation curves to GEA 3175 and NO. 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 x 10(-6) M) and LY 83583 (10(-6) M), the inhibitors of soluble guanylate cyclase, also reduced the relaxations induced by GEA 3175 and nitric oxide. However, ODQ did not affect salbutamol-evoked relaxation in the bovine small bronchioles. 5. GEA 3175-induced relaxations were reduced in potassium-rich (60 mmol l(-1) K+) solution. Glibenclamide (10(-6) M) markedly inhibited the relaxations induced by the opener of ATP-sensitive K+ channels, levcromakalim (3 x 10(-8)-10(-5) M), but it did not modify the relaxations induced by GEA 3175 or salbutamol. Apamin (5 x 10(-7) M), a blocker of the small Ca2+-activated K+-channels did not affect the relaxations to GEA 3175. In contrast, blockers of large Ca2+-activated K+-channels, charybdotoxin (3 x 10(-8)-10(-7) M) and iberiotoxin (10(-8) M), did inhibit the relaxations to GEA 3175. The combination of apamin and charybdotoxin did not induce an additional inhibitory effect on the relaxations to GEA 3175 compared to charybdotoxin alone. 6. In preparations where a concentration-response curve to GEA 3175 or NO was first obtained in the presence of LY 83583, incubation with charybdotoxin (10(-7) M) did produce an additional inhibitory effect of the relaxations. However. in the presence of ODQ (3 x 10(-6) M), iberiotoxin (10(-8) M) did not produce additional reduction of the NO- or GEA 3175-induced relaxations. 7. The present results suggest that the slow-releasing NO-donor GEA 3175 is more potent than the traditional NO donors in inducing relaxations of bovine bronchioles. GEA 3175, as for exogenously added NO, elicits relaxations through a cyclic GMP-dependent mechanism followed by opening of large conductance Ca2+-activated K+-channels.

Albuterol↗
PubMed1992

Nitric oxide and relaxation of pig lower urinary tract.

1. We studied the non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the L-arginine (L-ARG)/nitric oxide (NO) pathway in this response. 2. Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin-1 (ET-1), relaxed frequency-dependently in response to electrical stimulation. Maximum relaxation was obtained at 6-8 Hz, and amounted to 56 +/- 2%, 77 +/- 3% and 62 +/- 6% of the agonist-induced tension in preparations contracted by NA, carbachol, or ET-1, respectively. Exposure to NG-nitro-L-arginine (L-NOARG; 10(-7)-10(-5) M) concentration-dependently reduced the relaxant response in preparations contracted by NA. L-NOARG (10(-6) M) reduced the maximal response to 51 +/- 8% of control. L-NOARG (10(-5) M) abolished all relaxation, and unmasked a contractile component; D-NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET-1, L-NOARG (10(-5) M) markedly reduced relaxations evoked by electrical stimulation. 3. In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with L-ARG (10(-3) M), and the inhibitory effect of L-NOARG (10(-6) M) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies less than 6 Hz, but a small inhibition was observed at higher frequencies. 4. Administration of NO (present in acidified solution of NaNO2) induced concentration-dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET-1.L-NOARG (10-5 M) and L-ARG (10-3M) had no effect on these relaxations. However, methylene blue (10-S M) significantly shifted the concentration-response curve for NO to the right. NANC-relaxation and NO-induced relaxation of trigonal preparations were both inhibited by oxyhaemoglobin (10-5 M) and pyrogallol (10-4 M).5. In urethral preparations precontracted by NA, electrical stimulation caused frequency-dependent relaxations. A maximum relaxation of 73 +/- 4% was obtained at 10 Hz. Also in the urethra, NANCrelaxation was blocked by L-NOARG (10-5 M), and a contractile response generally appeared.6. Detrusor strips treated with alpha-beta methylene ATP (10-i M) and atropine (10-6 M), and then contracted by ET-1, showed relaxations (19 +/- 3% of the induced tension) in response to electrical field stimulation (2-20 Hz) only when the tension was high. No response at all, or small contractions, were found in response to electrical stimulation in K+ (35 mM)-contracted detrusor strips. Detrusor preparations contracted by carbachol were concentration-dependently relaxed by exogenously administered NO, SIN-1 (NO-donor), and isoprenaline, whereas vasoactive intestinal polypeptide had minor effects. NO and SIN-1 induced maximal relaxations of 63 +/- 3% and 70 +/- 4%, respectively, of the tension induced by carbachol. Isoprenaline produced an almost complete relaxation (96 +/- 4%).7. The results suggest that NANC-nerve mediated relaxation, involving the L-ARG/NO pathway, can be demonstrated consistently in the pig trigonal and urethral, but not in detrusor smooth muscle. The importance of this pathway for lower urinary tract physiology and pathophysiology remains to be established.

Animals↗
PubMed1998

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea.

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg9]-bradykinin and their relaxant mechanisms in the mouse isolated trachea. 2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg9]-bradykinin (each drug, 0.01-10 microM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01-10 microM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 microM). The relaxation induced by bradykinin was inhibited by the B2 receptor antagonist, D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (Hoe 140, 0.01-1 microM) in a concentration-dependent manner whereas the B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (0.01-1 microM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg9]-bradykinin (0.01-10 microM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg9-bradykinin was concentration-dependently inhibited by the B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (0.01-1 microM), whereas the B2 receptor antagonist, Hoe 140 (0.01-1 microM) was without effect. 3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01-1 microM), the relaxations induced by bradykinin and [des-Arg9]-bradykinin were inhibited concentration-dependently. 4. Two nitric oxide (NO) biosynthesis inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) and NG-nitro-L-arginine (L-NOARG, 100 microM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg9]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) inhibit the relaxations induced by bradykinin and [des-Arg9]-bradykinin. 5. Prostaglandin E2 (PGE2, 0.01-33 microM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 microM) and ODQ (10 microM) exerted no inhibitory effects on the relaxation induced by PGE2. 6. The NO-donor, sodium nitroprusside (SNP; 0.01-100 microM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1-1 microM) concentration-dependently inhibited the relaxation induced by SNP. 7. These data demonstrate that bradykinin and [des-Arg9]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B2-receptors and B1-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

Animals↗
PubMed1995

Effects of volatile anesthetics on acetylcholine-induced relaxation in the rabbit mesenteric resistance artery.

BACKGROUND: Vascular endothelium plays an important role in the regulation of vascular tone. Volatile anesthetics have been shown to attenuate endothelium-mediated relaxation in conductance arteries, such as aorta. However, significant differences in volatile anesthetic pharmacology between these large vessels and the small vessels that regulate systemic vascular resistance and blood flow have been documented, yet little is known about volatile anesthetic action on endothelial function in resistance arteries. Furthermore, endothelium-dependent relaxation mediated by factors other than endothelium-derived relaxing factor (EDRF) has recently been recognized, and there is no information available regarding volatile anesthetic action on non-EDRF-mediated endothelium-dependent relaxation. METHODS: Employing isometric tension recording and microelectrode methods, the authors first characterized the endothelium-dependent relaxing and hyperpolarizing actions of acetylcholine (ACh) in rabbit small mesenteric arteries, and tested the sensitivities of these actions to EDRF pathway inhibitors and K+ channel blockers. They then examined the effects of the volatile anesthetics isoflurane, enflurane, and sevoflurane on ACh-induced endothelium-dependent relaxation that was sensitive to EDRF inhibitors and that which was resistant to the EDRF inhibitors but sensitive to blockers of ACh-induced hyperpolarization. The effects of the volatile anesthetics on endothelium-independent sodium nitroprusside (SNP)-induced relaxation were also studied. RESULTS: Acetylcholine concentration-dependently caused both endothelium-dependent relaxation and hyperpolarization of vascular smooth muscle. The relaxation elicited by low concentrations of ACh (< or = 0.1 microM) was almost completely abolished by the EDRF inhibitors NG-nitro-L-arginine (LNNA), oxyhemoglobin (HbO2), and methylene blue (MB). The relaxation elicited by higher concentrations of ACh (> or = 0.3 microM) was only attenuated by the EDRF inhibitors. The remaining relaxation, as well as the ACh-induced hyperpolarization that was also resistant to EDRF inhibitors, were both specifically blocked by tetraethylammonium (TEA > or = 10 mM). Sodium nitroprusside, a NO donor, produced dose-dependent relaxation, but not hyperpolarization, in the endothelium-denuded (E[-]) strips, and the relaxation was inhibited by MB and HbO2, but not TEA (> or = 10 mM). One MAC isoflurane, enflurane, and sevoflurane inhibited both ACh relaxation that was sensitive to the EDRF inhibitors and the ACh relaxation resistant to the EDRF inhibitors and sensitive to TEA, but not SNP relaxation (in the E[-] strips). An additional finding was that the anesthetics all significantly inhibited norepinephrine (NE) contractions in the presence and absence of the endothelium or after exposure to the EDRF inhibitors. CONCLUSIONS: The results confirm that ACh has a hyperpolarizing action in rabbit small mesenteric resistance arteries that is independent of EDRF inhibitors but blocked by the K+ channel blocker TEA. The ACh relaxation in these resistance arteries thus appears to consist of distinct EDRF-mediated and hyperpolarization-mediated components. Isoflurane, enflurane, and sevoflurane inhibited both components of the ACh-induced relaxation in these small arteries, indicating a more global depression of endothelial function or ACh signaling in endothelial cells, rather than a specific effect on the EDRF pathway. All these anesthetics exerted vasodilating action in the presence of NE, the primary neurotransmitter of the sympathetic nervous system, which plays a major role in maintaining vasomotor tone in vivo. This strongly indicates that the vasodilating action of these anesthetics probably dominates over their inhibitory action on the EDRF pathway and, presumably, contributes to their known hypotensive effects in vivo. Finally, the vasodilating action of these anesthetics is, at least in part, independent from endothelium.

Acetylcholine↗
PubMed1994

Relaxation mechanisms induced by stimulation of nerves and by nitric oxide in sheep urethral muscle.

Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EFS) with stimulus-dependent non-adrenergic, non-cholinergic (NANC) relaxations. Exogenous nitric oxide (NO) (acidified NaNO2), S-nitroso-L-cysteine (NC), sodium nitroprusside (SNP), 8-Br-cGMP, dibutyryl-cAMP, forskolin and isoprenaline each relaxed precontracted transverse urethral preparations in a concentration-dependent manner in order of protency: NC > forskolin > isoprenaline = SNP > NO > 8-Br-cGMP = dibutyryl-cAMP. Longitudinally oriented preparations responded to NO and NC with concentration-dependent relaxation, no different from that observed in transverse strips. Methylene blue (MB) and oxyhaemoglobin (HbO2) each shifted the concentration-response curve for NO to the right without affecting EFS-induced relaxation. Similarly, concentration-dependent responses to NC were not affected by MB. The inhibition of relaxation to NO by MB was prevented by superoxide dismutase, suggesting the inhibition was caused by extracellular generation of superoxide anions. EFS-induced relaxation was accompanied by elevation of cGMP. However, for the same level of relaxation, exogenous NO and NC induced 15- and 23-times higher increases in cGMP values, respectively, than EFS. cAMP levels were not affected by EFS- or NO-induced relaxation, although a large increase accompanied relaxation induced by forskolin. Forskolin also increased cGMP content. Pretreatment with MB reduced basal levels of cGMP and inhibited both relaxation and rise in cGMP levels induced by NO. SNP-elicited relaxant responses, in the presence of MB, were accompanied by an accumulation of cGMP; cAMP levels were unaffected. MB reduced cGMP levels induced by NC, while the relaxant response was unchanged. In urethral preparations prelabelled with [3H]myoinositol, exposure to NA caused an accumulation of [3H]inositol phosphates, which was unaffected by pretreatment with 8-Br-cGMP or dibutyryl-cAMP. EFS failed to induce a relaxant response in excess [K+]o-contracted preparations, while relaxation with exogenous NO was unaffected. Ouabain abolished EFS-induced relaxation and reduced responses to NO. Neither TEA nor glibenclamide affected relaxation to either EFS or NO. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO2, K+ channel blockers (TEA and glibenclamide), ouabain or high [K+]o solution. This suggested that relaxation was caused by a mechanism independent of NO generation. A dense network of NADPH diaphorase-positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Salmeterol, formoterol, and salbutamol in the isolated guinea pig trachea: differences in maximum relaxant effect and potency but not in functional antagonism.

BACKGROUND: Formoterol and salmeterol are new long acting beta 2 adrenoceptor agonists. The maximum relaxant effect, potency and functional antagonism against carbachol induced contraction for salmeterol, formoterol and salbutamol have been compared in the guinea pig isolated trachea. In addition, the possibility of inducing a non-beta adrenoceptor mediated relaxation by salmeterol was studied. METHODS: Concentration response experiments were conducted with isolated tracheal preparations (n = 4-6 in all experiments), precontracted by carbachol to cause either 40% (60 nmol/l), 80% (0.3 mumol/l) or 100% (3 mumol/l, supramaximal) of the maximum contraction. Each beta agonist was added cumulatively at each level of precontraction. Additional cumulative concentration response experiments were conducted for salmeterol alone at the highest level of precontraction, with and without beta blockade by sotalol (1 mmol/l). With the drug concentrations which produced the maximum response and the highest level of precontraction, the relaxation of formoterol (10 nmol/l) and salmeterol (1 mumol/l) was also compared non-cumulatively. Finally, with the corresponding drug concentrations and precontraction, the relaxant effect was compared for formoterol (10 nmol/l) in salmeterol relaxed airways with that of salmeterol (1 mumol/l) in formoterol relaxed airways. RESULTS: The increase in carbachol concentration from 60 nmol/l to 3 mumol/l induced a rightward shift in the mean (SE) concentration (log steps) causing 50% maximum relaxation for salmeterol (0.73 (0.17)), formoterol (0.85 (0.18)), and salbutamol (1.13 (0.11)). Significant differences in the maximum relaxant effect were shown at the highest level of precontraction only, with a remaining active tension of percentage precontraction of 27% (4%) for 1 mumol/l salbutamol and 35% (3%) for 10 nmol/l formoterol compared with 50% (2%) for 1 mumol/l salmeterol. The rank order of potency was: formoterol > salbutamol approximately salmeterol at all levels of precontraction (-log EC50: 9.32 (0.05) for formoterol, 7.82 (0.08) for salbutamol, and 7.50 (0.13) for salmeterol at 80% maximum precontraction). Beta blockade by sotalol (1 mmol/l) significantly inhibited the relaxation induced by salmeterol (1 mumol/l) (remaining active tension: 104% (1%) v 71% (11%) of precontraction) but not the relaxation induced by salmeterol (10 mumol/l) (remaining active tension: 75% (5%) v 71% (12%) of precontraction). In the non-cumulative experiments, formoterol displayed more relaxant effect than salmeterol (remaining active tension: 51% (6%) v 65% (6%) of precontraction). Finally, formoterol significantly relaxed salmeterol relaxed airways (relaxant effect: 22% (8%) of precontraction) whereas there was no significant response to salmeterol in formoterol relaxed airways (relaxant effect: 5% (12%) of precontraction). CONCLUSIONS: In the guinea pig isolated trachea, formoterol and salbutamol produce more relaxant effect than salmeterol, suggesting that salmeterol is a partial beta 2 agonist. Very high concentrations of salmeterol may induce non-beta adrenoceptor mediated relaxation. Formoterol is more potent than both salbutamol and salmeterol. There is no pronounced difference in the magnitude of antagonism against carbachol induced contractions between salmeterol, formoterol, and salbutamol.

Adrenergic beta-Agonists↗
PubMed1995

Testosterone relaxes rabbit coronary arteries and aorta.

BACKGROUND: Until menopause, women appear to be protected from coronary heart disease. Evidence suggests that estrogen may play a role in the protection of the cardiovascular system by exerting a beneficial effect on risk factors such as cholesterol metabolism and by a direct effect on the coronary arteries. To date there has been no evidence linking testosterone with the occurrence of coronary heart disease. Testosterone may affect the cardiovascular system directly, thus partially explaining the difference in the incidence of coronary artery disease in men and premenopausal women. The purpose of this study was to assess the direct effect of testosterone and a number of testosterone analogues on rabbit coronary arteries and aorta in vitro. METHODS AND RESULTS: Rings of coronary artery and aorta of adult male or nonpregnant female New Zealand White rabbits were suspended in organ baths containing Krebs solution; isometric tension then was measured. The response to testosterone was investigated in prostaglandin F2 alpha (PGF 2 alpha)- and KCl-contracted rings. The effects of endothelium and nitric oxide synthase, prostaglandin synthetase, and guanylate cyclase inhibition on testosterone-induced relaxation were investigated. The effects of ATP-sensitive potassium channels and potassium conductance were also assessed. Relaxing responses in the presence of aromatase inhibition and testosterone receptor blockade were performed. The relaxing responses to the testosterone analogues etiocholan-3 beta-ol-17-one, epiandrosterone, 17 beta-hydroxy-5 alpha-androst-1-en-3-one, androst-16-en-3-ol, and testosterone enanthanate were measured. Testosterone relaxed rabbit coronary arteries and aorta. There was no significant difference between the relaxation effect of testosterone with or without endothelium. Similar results were obtained from male and nonpregnant female rabbits. The relaxing response of testosterone in the coronary artery was significantly greater than in the aorta. The relaxing response of testosterone in the coronary artery was significantly reduced by the potassium channel inhibitor barium chloride but not by the ATP-sensitive potassium channel inhibitor glibenclamide. The relaxing response to testosterone was greater in PGF 2 alpha-contracted rings compared with KCl-contracted rings. Inhibitors of nitric oxide synthase, prostaglandin synthetase, and guanylate cyclase did not affect relaxation induced by testosterone. Inhibition of aromatase and testosterone receptors did not affect relaxation. Testosterone did not shift the rabbit coronary arterial calcium concentration-dependent contraction curves, whereas verapamil did. There were, however, significant differences in the relaxing response to testosterone compared with testosterone analogues. Testosterone was the most potent relaxing agent, suggesting that there may be a structure-function relation in the relaxing response. CONCLUSIONS: Testosterone induces endothelium-independent relaxation in isolated rabbit coronary artery and aorta, which is neither mediated by prostaglandin I2 or cyclic GMP. Potassium conductance and potassium channels but not ATP-sensitive potassium channels may be involved partially in the mechanism of testosterone-induced relaxation. The in vitro relaxation is independent of sex and of a classic receptor. The coronary artery is significantly more sensitive to relaxation by testosterone than the aorta. Testosterone is a more potent relaxing agent of rabbit coronary artery than other testosterone analogues.

Animals↗
PubMed2001

Perinatal development influences mechanisms of bradykinin-induced relaxations in pulmonary resistance and conduit arteries differently.

OBJECTIVE: As bradykinin (BYK) relaxes conduit (EPA) and resistance (RPA) pulmonary arteries from both perinatal and adult lungs, we investigated whether this vasodilator's relaxation-mechanisms were altered during perinatal development, differed between EPA and RPA and differed with other endothelium-dependent vasodilators, acetyicholine (ACH) and substance P (SP). METHODS: Arteries from mature foetal (5 days), neonatal (approximately 5 min), newborn (60-84 h) and adult pigs (> or =6 months) were isolated, mounted for in vitro isometric force recording, activated with PGF(2alpha) (30 micromol/l) and relaxed with BYK (10 pmol/l-1 micromol/l), SP (10 pmol/l-0.1 micromol/l) or ACH (1 nmol/l-1 mmol/l). RESULTS: (i) BYK: L-NAME (100 micromol/l) attenuated relaxations in foetal EPA ( approximately 55%) but nearly abolished them in the adult ( approximately 80%). In RPA, L-NAME nearly abolished ( approximately 90%) relaxations in the foetus and this effect diminished progressively with age to approximately 20% in the adult. Indomethacin (IND, micromol/l) attenuated relaxations in neonatal (approximately 25%), new-born and adult EPA (both approximately 45%). Together, L-NAME and IND abolished relaxations in all EPA and in neonatal RPA but not in older RPA. SKF525a (100 micromol/l) attenuated relaxations in foetal RPA ( approximately 4%), diminishing in the adult RPA to approximately 10%. Together, SKF52Sa and L-NAME largely abolished relaxations in postnatal RPA (approximately 80%). Activation with K(+)=125 mmol/l attenuated relaxations in adult EPA (approximately 80%), foetal RPA ( approximately 45%) and neonatal RPA (approximately 75%) and abolished relaxations in RPA from older ages. (ii) ACH: L-NAME abolished relaxations in new-born EPA and RPA. In adult EPA, combined L-NAME and IND moderately attenuated relaxations. (iii) SP: Combined application of L-NAME and IND attenuated relaxations to a similar degree in new-born and adult EPA and RPA. CONCLUSIONS: In postnatal EPA, BYK-relaxations depend completely on prostaglandin- and NO-synthesis whereas those to SP (at all ages) and ACH (in the adult) do not. In RPA, BYK-relaxations develop from being completely dependant on the sole release of NO (foetus) to being almost completely independent of it (adult), a situation mimicked partially by SP but not by ACH, which, in new-born RPA is completely dependent on NO. BYK-relaxations in postnatal RPA depend on the release of a hyperpolarising factor generated through an SKF525a-sensitive pathway in conjunction with NO. The mechanisms of endothelium-dependent BYK-relaxations in the pulmonary vascular bed undergo diverging alterations, depending on the stage of development and arterial size/function. These changes are specific for BYK as they differ from those obtained from ACH or SP.

Acetylcholine↗
PubMed2005

ATP- and adenosine-induced relaxation of the smooth muscle of the pig urethra.

OBJECTIVES: To investigate relaxation mechanisms for ATP and adenosine in the pig urethra, together with the possible role of ATP in nerve-evoked urethral relaxations, as ATP is thought to cause bladder smooth muscle contraction via P2X receptors, whereas relaxation is mediated via G-protein coupled P2Y receptors, and ATP may also induce relaxation via breakdown to adenosine. MATERIALS AND METHODS: Circular muscle strips from the female pig urethra were mounted in tissue baths to record force; the effects of increasing concentrations of 1-300 microM ATP, the P2-receptor agonist 2-methylthioATP (2-MeSATP), adenosine, the stable adenosine-analogue, 5'(N-ethylcarboxamido) adenosine (NECA), ADP, uridine-triphosphate (UTP) and alpha,beta-methylene-ATP were assessed on the spontaneously developed tone. Responses to ATP were further assessed in the presence of G-protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS; 1-10 microM), the G-protein inhibitor guanosine 5'-O-(2-thio-diphosphate) (GDPbetaS; 10-100 microM), suramin (1-100 microM), the ecto-ATPase inhibitor 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine-5-triphosphate (ARL 67156, 10-100 microM), and the suggested P2Y receptor antagonist, reactive blue-2 (1-100 microM). The effect of the adenosine (P1) receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 1-100 microM) on responses to adenosine, and the effects of the adenosine reuptake inhibitor S(p-nitrobenzyl)-6-thioinosine (NBTI, 1-100 microM) on responses to adenosine and ATP were also assessed. Responses to electrical field stimulation (EFS, 12 and 30 Hz) in the presence of phentolamine (1 microM), scopolamine (1 microM) and N omega-nitro-L-arginine (0.3 mM) were studied before and after treatment with GTPgammaS, GDPbetaS, suramin, reactive blue-2 and ARL 67156. RESULTS: Strips were relaxed in a concentration-dependent manner by exogenously administered ATP and 2-meSATP, the relaxations being slowly developing and long-lasting. The relaxant effect evoked by both agonists at 300 microM amounted to about half of the spontaneously developed tone. The relaxation evoked by ATP was not significantly affected by GTPgammaS, GDPbetaS, suramin, ARL 67156 or reactive blue-2. Adenosine induced a concentration-dependent relaxation of the smooth muscle tone, reaching a maximum of approximately 70% at 300 microM, whereas 300 microM NECA only relaxed the preparations by approximately 35%. The adenosine-induced relaxation was not affected by treatment with 8-SPT. However, NBTI (1 microM) significantly reduced the relaxation evoked by 300 microM adenosine. ADP relaxed the smooth muscle tone by approximately 40% (300 microM). There was no response to UTP, and the effect of alpha,beta-methylene-ATP was negligible (5% relaxation at 100 microM). EFS caused slowly developing and long-lasting relaxations that were unaffected by GTPgammaS, GDPbetaS, suramin, reactive blue-2 and ARL 67156. CONCLUSIONS: These results suggest that exogenous ATP and adenosine relax the smooth muscle of the pig urethra in a manner similar to that evoked by electrical stimulation of nerves, although there was no evidence for involvement of a definable P2Y receptor subtype in these relaxations.

Adenosine↗
PubMed1996

Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery.

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutase (SOD; 30 u ml-1), potentiated relaxations to SIN-1 in all tissues, but did not alter the effects of charybdotoxin and ODQ and SIN-1-evoked relaxation. 7. These data show that although relaxation to the NO-donor SIN-1 is not significantly different between endothelium-intact and denuded arterial segments, the mechanisms which mediate SIN-1-evoked relaxation in the rat isolated mesenteric artery appear to be modulated by the basal release of endothelium-derived NO. In the presence of an intact endothelial cell layer, the major mechanism for SIN-1-evoked relaxation appears to be the activation of charybdotoxin-sensitive potassium channels. In contrast, when basal NO synthesis is inhibited, SIN-1 appears to cause full relaxation by both the activation of a charybdotoxin-sensitive pathway and the stimulation of soluble guanylyl cyclase.

Animals↗
PubMed1986

Mechanisms of histamine-induced relaxation in isolated monkey and dog coronary arteries.

Relaxations induced by histamine in helical strips of monkey coronary arteries were attenuated either by chlorpheniramine or cimetidine; the H1 antagonist suppressed the fast component of relaxation, whereas the H2 antagonist reduced the slow component. Combined treatment with these antagonists abolished the amine-induced relaxation. The relaxation was not influenced by indomethacin; however, the fast component of relaxation was inhibited by 5, 8, 11, 14-eicosatetraynoic acid, AA861, a lipoxygenase inhibitor, and methylene blue. In the arteries treated with methylene blue, relaxations were abolished by cimetidine. Removal of the endothelium reduced the relaxation markedly or reversed the relaxation to a contraction; chlorpheniramine reversed the contraction to a relaxation. In dog coronary arterial strips, histamine-induced relaxations were not attenuated by removal of the endothelium. Cimetidine shifted the dose-response curve for histamine to the right, but chlorpheniramine did not alter the response. Indomethacin, AA861 and methylene blue failed to inhibit the relaxation. The response of monkey coronary arteries to histamine appears to be a sum of the slight, persistent contraction, the transient relaxation and the slowly developing relaxation. The transient relaxation may be mediated by H1 receptors in the endothelium, the activation of which yields relaxing factor, resulting in an increase of cellular cyclic GMP in smooth muscle. The contraction and the slow relaxation appear to be associated with H1 and H2 receptors, respectively, in smooth muscle cells. Dog coronary arterial relaxations induced by histamine may be mediated exclusively by H2 receptors in muscle cell membrane.

Animals↗