Characterization of endothelium-derived relaxing factors released by bradykinin in human resistance arteries

1. Relaxing factors released by the endothelium and their relative contribution to the endothelium-dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries.
2. BK produced an endothelium-dependent relaxation of arteries pre-contracted with the thromboxane A₂ agonist, U46619. The B₂ receptor antagonist, Hoe 140 (0.1, 1 and 10 μM), produced a parallel shift to the right of the concentration-response curve to BK with a pA₂ of 7.75.
3. Neither the cyclo-oxygenase inhibitor, indomethacin (10 μM) alone, the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 300 μM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 μM) alone, nor the combination of L-NAME plus Hb affected the concentration-response curve to BK. Conversely, the combination of indomethacin with either L-NAME or Hb attenuated but did not abolish the BK-induced relaxation. By contrast, the relaxations produced by the Ca²⁺ ionophore, calcimycin ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187), and by the inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L-NAME. Also, the presence of indomethacin plus L-NAME produced contraction of arteries with functional endothelium.
4. The indomethacin plus L-NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L-NAME did not affect the nitric oxide donor, S-N-acetylpenicillamine-induced relaxation.
5. The indomethacin plus L-NAME resistant component of the relaxation to BK was significantly attenuated by the K⁺ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K⁺ channel blockers such as apamin (10 μM), 4-aminopyridine (100 μM), glibenclamide (10 μM), tetraethylammonium (10 mM) and charybdotoxin (50 nM).
6. In the presence of indomethacin plus L-NAME, the relaxation produced by BK was not affected by the phospholipase A₂ inhibitor, quinacrine (10 μM) or by the inhibitor of cytochrome P450, SKF 525a (10 μM). Another cytochrome P450 inhibitor, clotrimazole (10 μM) which also inhibits K⁺ channels, inhibited the relaxation to BK.
7. These results show that BK induces endothelium-dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo-oxygenase derived prostanoid(s) and another factor (probably an endothelium-derived hyperpolarizing factor). They indicate that nitric oxide and cyclo-oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P-450 pathway, in these arteries.

     

Endothelium-dependent sensory NANC vasodilatation: involvement of ATP,CGRP and a possible NO store

1. Non-adrenergic non-cholinergic (NANC) vasodilator nerves regulate tone in certain vascular beds. We have investigated the mechanisms of the NANC dilator response in the isolated small mesenteric artery of the rabbit by use of the tension myograph.
2. Small second or third order (150–300 μm in diameter) arteries of the rabbit mesenteric bed were mounted in a Mulvany tension myograph. Responses to electrical field stimulation (EFS) and exogenous vasodilators were investigated.
3. EFS (0.5–16 Hz, 10 V, 0.3 ms for 5 s), in the presence of guanethidine (5 μM) and atropine (1 μM) produced frequency-dependent relaxation of small arteries. Pretreatment with tetrodotoxin (1 μM) abolished the relaxation and desensitization with capsaicin (10 μM) strongly inhibited the relaxation.
4. Pretreatment with a P2Y-purinoceptor antagonist, basilen blue (3 μM) or a human calcitonin gene-related peptide (hCGRP) receptor antagonist, hCGRP_8–37 (1 μM) suppressed the NANC relaxation by approximately 40–60 % in each case and combined pretreatment almost abolished the relaxation.
5. The EFS-induced relaxation was suppressed by endothelium-removal, pretreatment with the soluble guanylyl cyclase inhibitor ODQ (1 μM) and the NO scavenger oxyhaemoglobin (OxyHb; 20 μM) but not by NO synthase inhibitors N^G-nitro-L-arginine methyl ester (L-NAME; 300 μM) or N^G-nitro-L-arginine (L-NOARG; 300 μM). Combined pretreatment with ODQ and CGRP_8–37 almost abolished the relaxation.
6. A P2Y-purinoceptor agonist, 2-methylthio ATP, produced endothelium-dependent relaxation which was inhibited by L-NAME and ODQ (1 μM), whilst hCGRP produced endothelium-independent and ODQ-insensitive relaxation.
7. Ultraviolet light (320 nm, 5 shots over 20 s) produced relaxation that was blocked by both OxyHb and ODQ but not by N^G-monomethyl-L-arginine (L-NMMA, 300 μM).
8. The present study suggests that EFS-induced NANC relaxation of the mesenteric small artery of the rabbit is mediated mainly by capsaicin-sensitive sensory C-fibres and that both ATP and CGRP are involved. The action of ATP released by EFS appears to be endothelium-dependent and involve activation of soluble guanylyl cyclase, but is resistant to inhibitors of NO synthase. The response to CGRP is endothelium-independent. These results show that ATP and CGRP account fully for the NANC relaxation of this vessel type and that the endothelium is involved in NANC-induced relaxation. The endothelium-dependent part of the response is consistent with the release of NO, either from NO synthase, incompletely inhibited by the NO synthase inhibitors, or by some preformed stores.

     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

Involvement of K+ channel permeability changes in the L-NAME and indomethacin resistant part of adenosine-5′-O-(2-thiodiphosphate)-induced relaxation of pancreatic vascular bed

1. We have previously demonstrated that adenosine-5′-O-(2-thiodiphosphate) (ADPβS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI₂) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na⁺/K⁺ pump, of ATP-sensitive K⁺ (K_ATP) channels and of small (SK_Ca) or large (BK_Ca) conductance Ca²⁺-activated K⁺ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase.
2. In control conditions, ADPβS (15 μM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N^ω-nitro-L-arginine methyl ester (L-NAME, 200 μM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPβS alone. The addition of indomethacin (10 μM) did not significantly modify the profile of ADPβS-induced vasodilatation.
3. Ouabain (100 μM) decreased basal pancreatic flow rate and did not modify ADPβS-induced relaxation. This inhibitor of the Na⁺/K⁺ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPβS vasodilatation.
4. The K_ATP inhibitor tolbutamide (185 μM) did not significantly modify basal pancreatic flow rate and ADPβS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPβS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPβS vasodilatation was not changed by the closure of K_ATP.
5. The SK_Ca inhibitor apamin (0.1 μM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK_Ca channels changed the one minute vasodilator effect of ADPβS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPβS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPβS-induced relaxation remained unchanged in the presence of apamin.
6. Charybdotoxin (0.2 μM), an inhibitor of BK_Ca, increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK_Ca channels reversed the one minute vasodilator effect of ADβPS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPβS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPβS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPβS infusion. The maximal vasodilator effect of ADPβS observed was not modified by the addition of apamin.
7. The results suggest that the L-NAME-resistant relaxation induced by ADPβS in the pancreatic vascular bed involves activation of BK_Ca, K_ATP and to a lesser extent of SK_Ca channels, but the L-NAME/indomethacin resistant part of ADPβS-induced relaxation is insensitive to the closure of K_ATP, SK_Ca and BK_Ca channels.

     

Differential actions of charybdotoxin on central and daughter branch arteries of the rabbit isolated ear

1. By use of rabbit isolated perfused intact ears and isolated perfused segments of central and first generation daughter branch ear arteries, we investigated the actions of charybdotoxin (ChTX), a blocker of calcium-activated K⁺ channels (K_Ca channels), and N^ω-nitro-L-arginine methyl ester (L-NAME) on pressure-flow and diameter-flow relationships.
2. ChTX (1 nM) induced an upwards shift in the pressure-flow curve in the rabbit intact isolated ear preconstricted with 5-hydroxytryptamine (5-HT; 100 nM) with subsequent administration of L-NAME (100 μM) inducing a further upwards shift. L-NAME itself induced an upwards shift in the pressure-flow curve, but subsequent administration of ChTX was without significant effect.
3. Microangiographic analysis revealed a tendency of ChTX (1 nM) to decrease vessel diameter in the central ear artery (G₀) with little effect on the first two generations of daughter branch arteries (G₁ and G₂) in the intact ear. Subsequent addition of L-NAME (100 μM) did not significantly further decrease vessel diameter in G₀, but did decrease vessel diameter in G₁ and G₂. L-NAME itself showed a tendency to decrease vessel diameter in G₀, G₁ and G₂ vessels with subsequent addition of ChTX being without significant effect.
4. In an isolated G₀ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) caused an upwards shift in the pressure-flow curve which was augmented by subsequent addition of L-NAME (100 μM). L-NAME (100 μM) itself caused an upwards shift in the pressure-flow curve but subsequent addition of ChTX (1 nM) had no significant effect.
5. In comparison, in an isolated G₁ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) had no significant effect on the pressure-flow curve relative to control, but subsequent addition of L-NAME (100 μM) caused an upwards shift. L-NAME (100 μM) itself induced an upwards shift in the pressure-flow curve with subsequent addition of ChTX (1 nM) being without significant effect.
6. ChTX (10 pM–10 nM) caused a concentration-dependent increase in perfusion pressure in isolated G₀ and G₁ preparations at fixed flow rates of 2 ml min⁻¹ and 0.5 ml min⁻¹, respectively. These responses were enhanced in the presence of L-NAME (100 μM) in G₁ but not G₀ preparations.
7. We conclude that at 1 nM, ChTX exhibits differential actions on central and daughter branch arteries of the intact ear of the rabbit, which are also apparent in the corresponding arteries when studied in isolation. The action of 1 nM ChTX in G₀ vessels may reflect inhibition of either the release or action of nitric oxide as it was blocked in the presence of L-NAME. At higher concentrations of ChTX, there would appear to be a direct constrictor effect on vascular smooth muscle which is apparent in both G₀ and G₁ vessels. This observed heterogeneity could reflect different distributions of K_Ca channels between central and daughter branch arteries at either the endothelial or smooth muscle levels, or both.

     

Potassium channel activation and relaxation by nicorandil in rat small mesenteric arteries

1. We used whole-cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.
2. Nicorandil (300 μM) activated currents with near-linear current-voltage relationships and reversal potentials near to the equilibrium potential for K⁺.
3. The nicorandil-activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.
4. One hundred μM nicorandil had no effect on currents through Ca²⁺ channels recorded in response to depolarizing voltage steps using 10 mM Ba²⁺ as a charge carrier. A small reduction in current amplitude was seen in 300 μM nicorandil, though this was not statistically significant.
5. In arterial rings contracted with 20 mM K⁺ Krebs solution containing 200 nM BAYK 8644, nicorandil produced a concentration-dependent relaxation with mean pD₂=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD₂=4.32±0.05), as did 60 mM K⁺. LY 83583 caused a dose-dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.
6. Metabolic inhibition with carbonyl cyanide m-chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.
7. We conclude that nicorandil activates K_ATP channels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.

     

Diazoxide- and leptin-activated KATP currents exhibit differential sensitivity to englitazone and ciclazindol in the rat CRI-G1 insulin-secreting cell line

1. The effects of the antidiabetic agent englitazone and the anorectic drug ciclazindol on ATP-sensitive K⁺ (K_ATP) channels activated by diazoxide and leptin were examined in the CRI-G1 insulin-secreting cell line using whole cell and single channel recording techniques.
2. In whole cell current clamp mode, the hyperglycaemic agent diazoxide (200 μM) and the ob gene product leptin (10 nM) hyperpolarised CRI-G1 cells by activation of K_ATP currents. K_ATP currents activated by either agent were inhibited by tolbutamide, with an IC₅₀ for leptin-activated currents of 9.0 μM.
3. Application of englitazone produced a concentration-dependent inhibition of K_ATP currents activated by diazoxide (200 μM) with an IC₅₀ value of 7.7 μM and a Hill coefficient of 0.87. In inside-out patches englitazone (30 μM) also inhibited K_ATP channel currents activated by diazoxide by 90.8±4.1%.
4. In contrast, englitazone (1–30 μM) failed to inhibit K_ATP channels activated by leptin, although higher concentrations (>30 μM) did inhibit leptin actions. The englitazone concentration inhibition curve in the presence of leptin resulted in an IC₅₀ value and Hill coefficient of 52 μM and 3.2, respectively. Similarly, in inside-out patches englitazone (30 μM) failed to inhibit the activity of K_ATP channels in the presence of leptin.
5. Ciclazindol also inhibited K_ATP currents activated by diazoxide (200 μM) in a concentration-dependent manner, with an IC₅₀ and Hill coefficient of 127 nM and 0.33, respectively. Furthermore, application of ciclazindol (1 μM) to the intracellular surface of inside-out patches inhibited K_ATP channel currents activated by diazoxide (200 μM) by 86.6±8.1%.
6. However, ciclazindol was much less effective at inhibiting K_ATP currents activated by leptin (10 nM). Ciclazindol (0.1–10 μM) had no effect on K_ATP currents activated by leptin, whereas higher concentrations (>10 μM) did cause inhibition with an IC₅₀ value of 40 μM and an associated Hill coefficient of 2.7. Similarly, ciclazindol (1 μM) had no significant effect on K_ATP channel activity following leptin addition in excised inside-out patches.
7. In conclusion, K_ATP currents activated by diazoxide and leptin show different sensitivity to englitazone and ciclazindol. This may be due to differences in the mechanism of activation of K_ATP channels by diazoxide and leptin.

     

Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries

1. In the presence of N^G-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 μM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery.
2. Inhibitors of adenosine 5′-triphosphate (ATP)-sensitive potassium (K)-channels (K_ATP; glibenclamide, 10 μM), voltage-sensitive K-channels (K_V; dendrotoxin-I, 0.1 μM or 4-aminopyridine, 1 mM), small (SK_Ca; apamin, 0.1 μM) and large (BK_Ca; iberiotoxin, 0.1 μM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine.
3. Synthetic charybdotoxin (0.1 μM), an inhibitor of BK_Ca and K_V, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was not significantly inhibited.
4. A combination of charybdotoxin (0.1 μM) and apamin (0.1 μM) abolished the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 μM) and apamin (0.1 μM).
5. Ciclazindol (10 μM), an inhibitor of K_V in rat portal vein smooth muscle, inhibited the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, and the relaxations were abolished when ciclazindol (10 μM) was combined with apamin (0.1 μM).
6. Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 μM) and apamin (0.1 μM), whereas each toxin had little effect alone.
7. The results suggest that K_V, but not K_ATP and BK_Ca, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both K_V and SK_Ca are activated by EDHF. However, a single type of K-channel, which may be structurally related to K_V and allosterically regulated by apamin, could also be the target for EDHF.

     

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 μM N^ω-nitro-L-arginine and 10 μM indomethacin.
2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H₁-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 μM).
3. The cytochrome P450 inhibitors proadifen (10 μM) and clotrimazole (10 μM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 μM) and 7-ethoxyresorufin (10 μM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 μM) and LEM (1 μM)-induced hyperpolarization.
4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 μM), glibenclamide (10 μM) and BaCl₂ (50 μM) had no significant effect on responses to ACh, AA and 11,12-EET.
5. IBTX (100 nM) significantly reduced both 11,12-EET (33 μM) and AA (30 μM) hyperpolarization without affecting the ACh (1 μM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations.
6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

     

Nitric oxide-related cyclic GMP-independent relaxing effect of N-acetylcysteine in lipopolysaccharide-treated rat aorta

1. We have recently demonstrated the formation of protein-bound dinitrosyl-iron complexes (DNIC) in rat aortic rings exposed to lipopolysaccharide (LPS) and shown that N-acetylcysteine (NAC) can promote vasorelaxation in these arteries, possibly via the release of nitric oxide (NO) as low molecular weight DNIC from these storage sites. The aim of the present study was to investigate further the mechanism of the relaxation induced by NAC in LPS-treated vessels.
2. In rings incubated with LPS (10 μg ml⁻¹ for 18 h) and precontracted with noradrenaline (NA, 3 μM) plus N^ω-nitro-L-arginine methylester (L-NAME, 3 mM), the relaxation evoked by NAC (0.1 to 10 mM) was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μM, a selective inhibitor of soluble guanylyl cyclase) but not affected by Rp-8-bromoguanosine 3′5′-cyclic monophosphorothioate (Rp-8BrcGMPS, 60 μM a selective inhibitor of cyclic GMP-dependent protein kinase). Tetrabutylammonium (TBA, 3 mM, as a non selective K⁺ channels blocker) or elevated concentration of external KCl (25 or 50 mM) significantly attenuated the NAC-induced relaxation. Selective K⁺ channels blockers (10 μM glibenclamide, 0.1 μM charybdotoxin, 0.5 μM apamin or 3 mM 4-aminopyridine) did not affect the NAC-induced relaxation. The relaxing effect of NAC (10 mM) was not associated with an elevation of guanosine 3′ : 5′ cyclic monophosphate (cyclic GMP) in LPS-treated rings.
3. In aortic rings precontracted with NA (0.1 μM), low molecular weight DNIC (with thiosulphate as ligand, 1 nM to 10 μM) evoked a concentration-dependent relaxation which was antagonized by ODQ (1 μM) and Rp-8BrcGMPS (150 μM) but not significantly affected by TBA (3 mM) or by the use of KCl (50 mM) as preconstricting agent. The relaxation produced by DNIC (0.1 μM) was associated with an 11 fold increase in aortic cyclic GMP content, which was completely abolished by ODQ (1 μM).
4. Taken together with our previous data, the main finding of the present study is that the vascular relaxation induced by NAC in LPS-treated aorta, although probably related to NO through an interaction via preformed NO stores, was not mediated by activation of the cyclic GMP pathway. It may involve the activation of TBA-sensitive K⁺ channels. The differences in the mechanism of relaxation induced by NAC and by exogenous DNIC suggest that the generation of low molecular weight DNIC from protein-bound species does not play a major role in the NAC-induced relaxation observed in LPS-treated rat aorta. In addition, it is suggested that ODQ may display other properties than the inhibition of soluble guanylyl cyclase.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Spatial heterogeneity in the mechanisms contributing to acetylcholine-induced dilatation in the rabbit isolated ear

1. Using an X-ray microangiographic technique in rabbit isolated perfused ears preconstricted with 5-HT (300 nM) and histamine (300 nM), we investigated the combined actions of N^ω-nitro-L-arginine methyl ester (L-NAME) and indomethacin on acetylcholine-induced depressor responses.
2. Under control conditions, acetylcholine (10 nM–30 μM) induced a concentration-dependent reversal of the pressor response, reaching a maximum of 66.0±13.6% (n=6). In the presence of L-NAME (300 μM) and indomethacin (10 μM), this depressor action was reduced, reaching a maximum of 38.6±5.9% (n=6).
3. The control response was associated with substantial vasodilatation in the central ear artery (G₀), a smaller dilatory action on first generation branch arteries (G₁) and no effect on second generation branch arteries (G₂). In the presence of L-NAME and indomethacin, vasodilatation occurred in G₂ with no effect in G₀ or G₁.
4. Two calcium-activated K⁺ channels blockers, charybdotoxin (ChTX; 10 nM) and penitrem A (100 nM), further inhibited, but did not abolish, the L-NAME- and indomethacin-resistant response to acetylcholine (10 nM–300 μM). Both agents abolished the vasodilatory action of acetylcholine in G₂.
5. In conclusion, L-NAME and indomethacin induced a shift in acetylcholine-induced vasodilatation from G₀ and G₁ to G₂. This is consistent with the suggestion that nitric oxide dominates in larger vessels whilst other mechanisms dominate in smaller vessels. The L-NAME- and indomethacin-resistant component was inhibited by ChTX and penitrem A, suggesting it is mediated, at least in part, by activation of K_Ca channels and could therefore involve a hyperpolarising mediator such as endothelium-derived hyperpolarising factor.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

KATP-channel activation: effects on myocardial recovery from ischaemia and role in the cardioprotective response to adenosine A1-receptor stimulation

1. Optimization of myocardial energy substrate metabolism improves the recovery of mechanical function of the post-ischaemic heart. This study investigated the role of K_ATP-channels in the regulation of the metabolic and mechanical function of the aerobic and post-ischaemic heart by measuring the effects of the selective K_ATP-channel activator, cromakalim, and the effects of the K_ATP-channel antagonist, glibenclamide, in rat fatty acid perfused, working hearts in vitro. The role of K_ATP channels in the cardioprotective actions of the adenosine A₁-receptor agonist, N⁶-cyclohexyladenosine (CHA) was also investigated.
2. Myocardial glucose metabolism, mechanical function and efficiency were measured simultaneously in hearts perfused with modified Krebs-Henseleit solution containing 2.5 mM Ca²⁺, 11 mM glucose, 1.2 mM palmitate and 100 mu l⁻¹ insulin, and paced at 300 beats min⁻¹. Rates of glycolysis and glucose oxidation were measured from the quantitative production of ³H₂O and ¹⁴CO₂, respectively, from [5-³H/U-¹⁴C]-glucose.
3. In hearts perfused under aerobic conditions, cromakalim (10 μM), CHA (0.5 μM) or glibenclamide (30 μM) had no effect on mechanical function. Cromakalim did not affect glycolysis or glucose oxidation, whereas glibenclamide significantly increased rates of glycolysis and proton production. CHA significantly reduced rates of glycolysis and proton production but had no effect on glucose oxidation. Glibenclamide did not alter CHA-induced inhibition of glycolysis and proton production.
4. In hearts reperfused for 30 min following 30 min of ischaemia, left ventricular minute work (LV work) recovered to 24% of aerobic baseline values. Cromakalim (10 μM), administered 5 min before ischaemia, had no significant effect on mechanical recovery or glucose metabolism. CHA (0.5 μM) significantly increased the recovery of LV work to 67% of aerobic baseline values and also significantly inhibited rates of glycolysis and proton production. Glibenclamide (30 μM) significantly depressed the recovery of mechanical function to <1% of aerobic baseline values and stimulated glycolysis and proton production.
5. Despite the deleterious actions of glibenclamide per se in post-ischaemic hearts, the beneficial effects of CHA (0.5 μM) on the recovery of mechanical function and proton production were not affected by glibenclamide.
6. The data indicate that the cardioprotective mechanism of adenosine A₁-receptor stimulation does not involve the activation of K_ATP-channels. Furthermore, in rat fatty acid perfused, working hearts, stimulation of K_ATP-channels is not cardioprotective and has no significant effects on myocardial glucose metabolism.

     

Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres

1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade.
2. Tityus serrulatus venom (3–100 μg), acetylcholine (ACh; 0.3–30 nmol) and glyceryl trinitrate (GTN; 0.5–10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 μM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 μM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 μM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible.
3. The non-selective NO synthase (NOS) inhibitors N^Ω-nitro-L-arginine methyl ester (L-NAME; 10 μM) and N^G-iminoethyl-L-ornithine (L-NIO; 30 μM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 μM), but not D-arginine (300 μM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 μM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [³H]-L-arginine to [³H]-L-citrulline.
4. The protease inhibitor aprotinin (Trasylol; 10 μg ml⁻¹) and the bradykinin B₂ receptor antagonist Hoe 140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K⁺ channel antagonist glibenclamide (10 μM) and the Ca²⁺-activated K⁺  channel antagonists apamin (0.1 μM) and charybdotoxin (0.1 μM) also failed to affect the venom-induced relaxations. Similarly, the K⁺ channel blocker tetraethylammonium (TEA; 10 μM) had no effect on the venom-induced relaxations.
5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 μM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 μM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum.
6. The sodium channel blocker tetrodotoxin (TTX; 1 μM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 μM) also promptly reversed the response to the venom when infused during the relaxation phase.
7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 μM) or L-NAME (10 μM).
8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.

     

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.
2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μM). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).
3. L-Nitro-arginine-methyl ester (L-NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2–2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.
4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine.
5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced.
6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME. L-Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.
7. S-methyl-L-thiocitrulline (10 μM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM).
8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

     

S-nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation

1. The effects of the antioxidants ascorbic acid and α-tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S-nitrosothiols, authentic nitric oxide (NO) and nitrergic non-adrenergic non-cholinergic stimulation of the rat gastric fundus.
2. The S-nitrosothiols S-nitrosocysteine (1–100 nM), S-nitrosoglutathione (0.01–3 μM) and S-nitroso-N-acetylpenicillamine (0.01–3 μM) induced concentration-dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F_2α. The relaxations to all S-nitrosothiols were concentration-dependently enhanced by the antioxidants ascorbic acid (0.1–3 μM) and α-tocopherol (3–30 μM) and inhibited by the metal chelator EDTA (26 μM).
3. Ascorbic acid and α-tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 μM) or α-tocopherol (1 μM) were injected in the organ bath 1 minute after S-nitrosoglutathione (0.1 μM) or after S-nitroso-N-acetylpenicillamine (0.1 μM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 μM). Such a relaxation to ascorbic acid or α-tocopherol was not observed in the presence of S-nitrosocysteine (10 nM).
4. Electrical field stimulation (0.5–4 Hz) of the precontracted rat gastric fundus strips induced frequency-dependent nitrergic relaxations which were mimicked by authentic NO (3–300 nM) and by acidified sodium nitrite NaNO₂ (0.3–10 μM). Ascorbic acid (0.3–3 μM), α-tocopherol (3–30 μM) or EDTA (26 μM) did not affect the relaxations to nitrergic stimulation, NO or NaNO₂.
5. In summary, relaxations to S-nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and α-tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S-nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S-nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

     

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 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). 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 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) 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 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) 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 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) 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.

     

The actions of the cannabinoid receptor antagonist,SR 141716A,in the rat isolated mesenteric artery

1. The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 μM, it caused concentration-dependent, but endothelium-independent, relaxations of both methoxamine- and 60 mM KCl-precontracted vessels.
2. SR 141716A (at 10 μM, but not at 1 μM) inhibited contractions to Ca²⁺ in methoxamine-stimulated mesenteric arteries previously depleted of intracellular Ca²⁺ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca²⁺.
3. SR 141716A (10 μM) caused a 130 fold rightward shift in the concentration-response curve to levcromakalim, a K⁺ channel activator, but had no effect at 1 μM.
4. SR 141716A (10 μM) attenuated relaxations to NS 1619 (which activates large conductance, Ca²⁺-activated K⁺ channels; BK_Ca). The inhibitory effect of SR 141716A on NS 1619 was not significantly different from, and was not additive with, that caused by a selective BK_Ca inhibitor, iberiotoxin (100 nM). SR 141716A (1 μM) did not effect NS 1619 relaxation.
5. SR 141716A (10 μM) had no effect on relaxations to the nitric oxide donor S-nitroso-N-acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl.
6. The results show that, at concentrations of 10 μM and above, SR 141716A causes endothelium-independent vasorelaxation by inhibition of Ca²⁺ entry. It also inhibits relaxations mediated by K⁺ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor-dependent processes.

     

NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery

1. The nature and cellular mechanisms that are responsible for endothelium-dependent relaxations resistant to indomethacin and N^G-nitro-L-arginine methyl ester (L-NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit.
2. In the presence of the cyclo-oxygenase inhibitor, indomethacin (10 μM), acetylcholine (ACh) induced a concentration- and endothelium-dependent relaxation of PE-induced tone which was more potent than the calcium ionophore A23187 with pD₂ values of 7.03±0.12 (n=8) and 6.37±0.12 (n=6), respectively. The ACh-induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD₂ value 7.00±0.10 and maximal relaxation 99±3%, n=6). Bradykinin and histamine (0.01–100 μM) had no effect either upon resting or PE-induced tone (n=5).
3. In the presence of indomethacin plus the NO synthase inhibitor, L-NAME (30 μM), the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD₂ value and the maximal relaxation decreasing to 6.48±0.10 and 67±3%, respectively (for both P<0.01, n=8). The L-NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n=8).
4. The Ca²⁺-activated K⁺ (K_Ca) channel blockers, tetrabutylammonium (TBA, 1 mM, n=5) and charybdotoxin (CTX, 0.1 μM, n=5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI₂)-independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 M, n=8) or apamin (1–3 μM, n=6) only partially inhibited the relaxation.
5. Inhibitors of the cytochrome P450 mono-oxygenase, SKF-525A (1–10 μM, n=6), clotrimazole (1 μM, n=5) and 17-octadecynoic acid (17-ODYA, 3 μM, n=7) also reduced the NO/PGI₂-independent relaxation response to ACh.
6. In endothelium-denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either K_Ca channel blockade with apamin (1 μM, n=5) or CTX (0.1 μM, n=5), or by the cytochrome P450 mono-oxygenase blockers SKF-525A (10 μM, n=4) and clotrimazole (10 μM, n=5). However, the NO-induced response was shifted to the right by LY83583 (10 μM, n=4), a guanylyl cyclase inhibitor, with the pD₂ value decreasing from 6.95±0.14 to 6.04±0.09 (P<0.01).
7. ACh (0.01–100 μM) induced a concentration-dependent relaxation of PE-induced tone in endothelium-denuded arterial segments sandwiched with endothelium-intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 μM) plus L-NAME (30 μM), but abolished by the combination of indomethacin, L-NAME and TBA (1 mM, n=5).
8. These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450-derived arachidonic acid metabolite, and (c) EDHF-induced relaxation involves the opening of at least two types of K_Ca channels, whereas NO mediates vasorelaxation via a guanosine 3′: 5′-cyclic monophosphate (cyclic GMP)-mediated pathway, in which a cytochrome P450 pathway and K_Ca channels do not seem to be involved.