Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea.
2. The selective 5-HT₂ receptor antagonist ketanserin (0.1 μM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA).
3. The partial agonist for 5-HT_1A receptors, 8-OH-DPAT (1 μM), and the full agonist for 5-HT₁ receptors, 5-CT (0.3 μM), potentiated the submaximal contractions induced by the 5-HT₂ receptor agonist α-methyl-5-HT (0.1 μM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 μM), as well as the 5-HT_1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 μM each), caused significant inhibition of the tracheal contractions induced both by NKA (10  nM–3  μM) and 5-HT (10 nM–10 μM) (n=4–10). This suggests that activation of 5-HT_1A receptors potentiates the 5-HT₂ receptor-mediated contractions.
4. SDZ 216525 (0.1 μM) significantly reduced the maximal contraction produced by 1 μM NKA (n=10, P<0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT_1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells.
5. Even in the presence of atropine (1 μM), 8-OH-DPAT (1 μM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 μM), SDZ 216525 (0.1 μM), NAN-190 (0.1 μM) and 8-OH-DPAT (1 μM) (n=4–6). These data demonstrate that the 5-HT_1A receptor-mediated potentiation of contractile responses is not due to non-specific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation.
6. The selective 5-HT_1B/1D receptor antagonist GR 127935, the selective 5-HT₃ receptor antagonist tropisetron and the selective 5-HT₄ receptor antagonists SB 204070 and GR 113808 (0.1 μM each) had no effect on the concentration-response curve for NKA (n=6–10), ruling out the involvement of 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors.
7. The α-adrenoreceptor antagonist phentolamine (1 μM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of α-adrenoreceptors.
8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT₂ receptors. Activation of 5-HT_1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT₂ receptor activation. The 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors are not involved in the NKA-induced contraction of rat airways.

     

Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA).
2. In endothelium-intact strips, histamine (His, 3–10 μM) and high K⁺ (20–80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 μM), 5-hydroxytryptamine (10 μM) and 9,11-epithio-11, 12-methano-thromboxane A₂ (10 nM) each produced only a small contraction (less than 5% of the maximum K⁺-induced contraction).
3. N^G-nitro-L-arginine (L-NOARG, 100 μM), but not indomethacin (10 μM), greatly enhanced the phasic and the tonic contractions induced by His (1–10 μM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3–3 μM) caused concentration-dependent relaxation (maximum relaxation by 89.7±7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 μM), but not glibenclamide (3 μM), abolished the relaxation induced by ACh (0.3–3 μM) in L-NOARG-treated strips (n=4, P<0.05).
4. In endothelium-intact tissues, His (3 μM) depolarized the smooth muscle membrane potential (by 4.4±1.8 mV, n=12, P<0.05) whereas ACh (3 μM) caused membrane hyperpolarization (−20.9±3.0 mV, n=25, P<0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (−23.5±5.9 mV, n=8, P<0.05) or glibenclamide (−20.6±5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to −5.8±3.2 mV, n=3, P<0.05).
5. Sodium nitroprusside (0.1–10 μM) did not hyperpolarize the smooth muscle cell membrane potential (0.2±0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n=4, P<0.05).
6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.

     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

1. The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT_3A(b)) heterologously expressed in Xenopus laevis oocytes.
2. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC₅₀=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV.
3. The response to 5-HT was mimicked by the 5-HT₃ receptor selective agonists 2-methyl-5-HT (EC₅₀=4.1±0.2 μM), 1-phenylbiguanide (EC₅₀=3.0±0.1 μM), 3-chlorophenylbiguanide (EC₅₀=140± 10 nM), 3,5-dichlorophenylbiguanide (EC₅₀=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC₅₀= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT.
4. Responses evoked by 5-HT at EC₅₀ were blocked by the 5-HT₃ receptor selective antagonist ondansetron (IC₅₀=231±22 pM) and by the less selective agents (+)-tubocurarine (IC₅₀=31.9± 0.01 nM) and cocaine (IC₅₀=2.1±0.2 μM).
5. The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT_3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT₃ receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT_3A subunit orthologues.

     

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT; 1 nM–100 μM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC₅₀; 68–84 nM) was more sensitive than the longitudinal muscle (EC₅₀; 1.3–1.44 μM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations.
2. Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L-NAME (100 μM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells.
3. 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, α-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.
4. Inhibition by 5-HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA₂ values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).
5. Ro 20-1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5-HT.
6. 5-HT (1 nM–1 μM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle.
7. From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT₇ receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT₇ receptors.

     

Investigation into the contractile response of melatonin in the guinea-pig isolated proximal colon: the role of 5-HT4 and melatonin receptors

1. The interaction of melatonin (N-acetyl-5-methoxytryptamine) with 5-hydroxytryptamine₄ (5-HT₄) receptors and/or with melatonin receptors (ML₁, ML₂ sites) has been assessed in isolated strips of the guinea-pig proximal colon. In the same preparation, the pharmacological profile of a series of melatonin agonists (2-iodomelatonin, 6-chloromelatonin, N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT)) was investigated.
2. In the presence of 5-HT_1/2/3 receptor blockade with methysergide (1 μM) and ondansetron (10 μM), melatonin (0.1 nM–10 μM), 5-HT (1 nM–1 μM) and the 5-HT₄ receptor agonist, 5-methoxytryptamine (5-MeOT: 1 nM–1 μM) caused concentration-dependent contractile responses. 5-HT and 5-MeOT acted as full agonists with a potency (−log EC₅₀) of 7.8 and 8.0, respectively. The potency value for melatonin was 8.7, but its maximum effect was only 58% of that elicited by 5-HT.
3. Melatonin responses were resistant to atropine (0.1 μM), tetrodotoxin (0.3 μM), and to blockade of 5-HT₄ receptors by SDZ 205,557 (0.3 μM) and GR 125487 (3, 30 and 300 nM). The latter antagonist (3 nM) inhibited 5-HT-induced contractions with an apparent pA₂ value of 9.6. GR 125487 antagonism was associated with 30% reduction of the 5-HT response maximum. Contractions elicited by 5-HT were not modified when melatonin (1 and 10 nM) was used as an antagonist.
4. Like melatonin, the four melatonin analogues concentration-dependently contracted colonic strips. The rank order of agonist potency was: 2-iodomelatonin (10.8) >6-chloromelatonin (9.9) ⩾ N-acetyl-5-HT (9.8) ⩾5-MCA-NAT (9.6) >melatonin (8.7), an order typical for ML₂ sites. In comparison with the other agonists, 5-MCA-NAT had the highest intrinsic activity.
5. The melatonin ML_1B receptor antagonist luzindole (0.3, 1 and 3 μM) had no effect on the concentration-response curve to melatonin. Prazosin, an α-adrenoceptor antagonist possessing moderate/high affinity for melatonin ML₂ sites did not affect melatonin-induced contractions at 0.1 μM. Higher prazosin concentrations (0.3 and 1 μM) caused a non-concentration-dependent depression of the maximal response to melatonin without changing its potency. Prazosin (0.1 and 1 μM) showed a similar depressant behaviour towards the contractile responses to 5-MCA-NAT.
6. In the guinea-pig proximal colon, melatonin despite some structural similarity with the 5-HT₄ receptor agonist 5-MeOT, does not interact with 5-HT₄ receptors (or with 5-HT_1/2/3 receptors). As indicated by the rank order of agonist potencies and by the inefficacy of luzindole, the most likely sites of action of melatonin are postjunctional ML₂ receptors. However, this assumption could not be corroborated with the use of prazosin as this ‘ML₂ receptor antagonist'' showed only a non-concentration-dependent depression of the maximal contractile response to both melatonin and 5-MCA-NAT. Further investigation with the use of truly selective antagonists at melatonin ML₂ receptors is required to clarify this issue.

     

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.

     

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.

     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

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.

     

Antagonist properties of (?)-pindolol and WAY 100635 at somatodendritic and postsynaptic 5-HT1A receptors in the rat brain

1. The aim of the present work was to characterize the 5-hydroxytryptamine_1A (5-HT_1A) antagonistic actions of (−)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT_1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices.
2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT_1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (−)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 μM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (−)-Pindolol also prevented the 5-HT_1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 μM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT₄ receptors persisted in the presence of 3 μM (−)-pindolol.
3. In the hippocampus, (−)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC₅₀=92 nM; apparent K_B=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC₅₀=522 nM; apparent K_B=115.1 nM). The block by (−)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action.
4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (−)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC₅₀=598 nM; apparent K_B=131.7 nM) or 100 nM ipsapirone (IC₅₀=132.5 nM; apparent K_B=124.9 nM). The effect of (−)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 μM (−)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT.
5. In vivo labelling of brain 5-HT_1A receptors by i.v. administration of [³H]-WAY 100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (−)-pindolol. (−)-Pindolol (15 mg kg⁻¹) injected i.p. either subchronically (2 day-treatment before i.v. injection of [³H]-WAY 100635) or acutely (20 min before i.v. injection of [³H]-WAY 100635) markedly reduced [³H]-WAY 100635 accumulation in all 5-HT_1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (−)-pindolol to prevent [³H]-WAY 100635 accumulation in the DRN and the CA1 and CA3 hippocampal areas.
6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC₅₀=4.9 nM, apparent K_B=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC₅₀=0.80 nM, apparent K_B=0.28 nM).

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

1. Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 μM), produced a slowly-developing contraction (`on'' response) followed by a quick phasic `off '' contraction (`off peak'' response) and a tonic response (`off late'' response), averaging 16±2, 73±3 and 20±4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 μM; 15 min before) abolished the overall response to EFS (n=8).
2. Neither in vitro capsaicin pretreatment (10 μM for 15 min), nor guanethidine (3 μM, 60 min before) affected the excitatory response to EFS (n=5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. N^ω-nitro-L-arginine (L-NOARG, 100 μM, 60 min before) or naloxone (10 μM, 30 min before) significantly enhanced the `on'' response (294±56 and 205±25% increase, respectively; n=6–8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655±90% increase of the `on'' component, n=6, P<0.05).
3. The tachykinin NK₂ receptor-selective antagonist MEN 11420 (1 μM) almost abolished both the `on'' and `off late'' responses (P<0.01; n=5 each) to EFS, and reduced the `off-peak'' contraction by 55±8% (n=5, P<0.01). The subsequent administration of the tachykinin NK<sub>1</sub> receptor-selective antagonist GR 82334 (1 μM) and of the tachykinin NK<sub>3</sub> receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 μM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 μM, GR 82334 significantly reduced (by 68±9%, P<0.05, n=6) the `on'' response to EFS.
4. The contractile `off peak'' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 μM each) was abolished (P<0.01; n=6) by the administration of the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 30 μM). PPADS (30 μM) selectively blocked (75±9 and 50±7% inhibition, n=4 each) the contractile responses produced by 100 and 300 μM ATP.
5. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed.
6. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK₂ (and possibly NK₁) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

Comparison of antioxidant activities of aminoguanidine,methylguanidine and guanidine by luminol-enhanced chemiluminescence

1. The objective of this study was to investigate the ability of aminoguanidine, methylguanidine and guanidine to inhibit free radicals or metabolites generated by either stimulated human leucocytes or cell-free systems using luminol-enhanced chemiluminescence (CL).
2. Aminoguanidine (0.1 μM–10 mM), methylguanidine (10 μM–10 mM) and guanidine (10 μM–10 mM) produced concentration-dependent inhibition (96±0.1%, n=7, 59±1.3%, n=6, and 62±3%, n=6, P<0.05 at 10 mM, respectively) in FMLP-stimulated leucocytes CL.
3. In cell-free experiments, hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), hydroxyl radical and peroxynitrite-induced CL responses were initiated by hydrogen peroxide (3.5 mM), NaOCl (50 μM), FeSO₄ (40 nM) and peroxynitrite (20 nM), respectively. Aminoguanidine, methylguanidine and guanidine produced concentration-dependent inhibition in H₂O₂-(69±0.7%, n=7, 26±1%, n=6, and 15±0.5%, n=6, at 1 mM, respectively) and HOCl-(84±0.3%, n=6, 50±1%, n=6, and 29±1%, n=7, at 1 mM, respectively) induced luminol CL. Peroxynitrite-induced CL was markedly attenuated in a concentration-dependent manner by aminoguanidine (99±0.1%, n=6, at 10 mM), methylguanidine (5±0.2%, n=6, at 10 mM) and guanidine (27±0.4%, n=7, at 10 mM). However, inhibition with aminoguanidine was found to be more marked than with methylguanidine and guanidine. Aminoguanidine (95±0.5%, n=6, at 1 mM) and methylguanidine (25±1%, n=6, at 1 mM), but not guanidine (2±1%, n=6, at 1 mM), significantly decreased ferrous iron-induced CL.
4. Collectively, these data suggest that aminoguanidine and a high concentration (⩾0.1 mM) of methylguanidine have direct scavenging activities against H₂O₂, HOCl, hydroxyl radical and peroxynitrite. Guanidine, at a high concentration (⩾0.1 mM), scavenges H₂O₂, HOCl and peroxynitrite, but not the hydroxyl radical. These direct scavenging properties may contribute to inhibitory effects of these compounds on human leucocyte CL.

     

Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A₁ receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area.
2. The concentration-response curve for the inhibitory effects of the selective adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC₅₀=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC₅₀=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope.
3. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non-selective mGluR agonist (which activates both group I and II mGluR), (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; 100 μM). L-CCG-I had no consistent effects and ACPD (100 μM) decreased by 19.4±1.8% (n=4) the fe.p.s.p. slope.
4. The concentration-response curve for the inhibitory effects of CPA (2–100 nM) on the fe.p.s.p. slope (EC₅₀=8.2 (6.9–9.6) nM) was displaced to the right by the group III mGluR selective agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 25 μM) (EC₅₀=17.7 (13.1–21.9) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by L-AP4 (25 μM) was blocked in the presence of the mGluR antagonist (selective for the group III mGluR), (R,S)-α-methyl-4-phosphonophenylglycine (MPPG; 200 μM).
5. Both the direct effect of DHPG on synaptic transmission and the attenuation of the inhibitory effect of CPA (10 nM) were prevented in the presence of the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C.
6. The attenuation by L-AP4 (25 μM) of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope was also prevented by the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C. But this effect seemed to be distinct from the direct effect of L-AP4 (25 μM) on synaptic transmission, which was not modified by the protein kinase C selective inhibitors.
7. We conclude that agonists of metabotropic glutamate receptors (Groups I and III) are able to attenuate the inhibitory effects of adenosine A₁ receptor activation in the hippocampus. This interaction may have pathophysiological relevance in hypoxia, in which there is marked release of both excitatory amino acids and the important endogenous neuroprotective substance, adenosine.

     

Hypoxic dilatation of porcine small coronary arteries: role of endothelium and KATP-channels

1. The aim of the present study was to determine the cellular mechanims and potential mediators involved in hypoxic dilatation of porcine small coronary arteries.
2. Small coronary arteries were isolated from a branch of the left anterior descending artery of porcine hearts, cannulated with glass micropipettes and studied in a perfusion myograph system. At a transmural pressure of 40 mmHg, the arteries had an internal diameter of 167.8±6.6 μm (n=37).
3. In arteries contracted with acetylcholine (ACh), hypoxia (0% O₂, 30 min) caused dilatation (86.9±6.7% relaxation, n=6) in vessels with endothelium but constriction in endothelium-denuded vessels.
4. Hypoxic vasodilatation occurring in arteries with endothelium was abolished by the K_ATP channel inhibitor, glibenclamide (0.44 μM), but was not affected by inhibition of nitric oxide synthase (L-NAME, 44 μM) or cyclo-oxygenase (indomethacin, 4.4 μM).
5. Bradykinin evoked endothelium-dependent relaxation that was inhibited by L-NAME (44 μM) but not glibenclamide 0.44 μM). Cromakalim (0.1–0.3 μM), a K_ATP channel opener, caused relaxation that was inhibited by glibenclamide, but was not affected by L-NAME (44 μM) and/or indomethacin (4.4 μM).
6. Endothelium-removal inhibited vasodilatation evoked by cromakalim, but increased vasodilator responses to the NO donor, SIN-1 (10⁻⁸ to 10⁻⁵ M).
7. These results indicate that hypoxia acted directly on vascular smooth muscle of small coronary arteries to cause contraction. However, this effect was overwhelmed by endothelium-dependent relaxation in response to hypoxia. This relaxation was most likely mediated by release of an endothelium-derived factor, distinct from nitric oxide or prostacyclin, that activated smooth muscle K_ATP-channels.

     

Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for up-regulation of endothelin-converting enzyme activity in disease

Overproduction of the potent vasoconstrictor peptide endothelin-1 (ET-1) is implicated in the pathogenesis of coronary artery disease. In endothelium-denuded human coronary arteries the response to big ET-1 was significantly enhanced in atherosclerotic arteries (coronary artery disease, CAD; n=7) with an EC₅₀ value of 96 nM (57–161 nM, 95% C.I.) compared to 274 nM (205–365 nM) in non-diseased arteries (dilated cardiomyopathy, DCM; n=10) (Mann-Whitney U-test, P<0.05). Higher levels of immunoreactive endothelin (ET) could be detected by radioimmunoassay in bathing medium taken from CAD arteries than from DCM arteries (2.8±0.5 nM, n=5 vs 1.1±0.2 nM, n=7) (Student''s two-tailed t-test, P<0.05). There were no differences in responses of arteries from either group to ET-1 (EC₅₀ 10 nM, CAD vs 14 nM, DCM). The enhanced response of atherosclerotic human coronary arteries to big ET-1 appears to be due to up-regulation of endothelin-converting enzyme (ECE) activity rather than to an augmented response of the arteries to ET-1. This non-endothelial ECE may therefore be an important therapeutic target in coronary artery disease.     

Stretch-evoked inhibition of spontaneous migrating contractions in a whole mount preparation of the guinea-pig upper urinary tract

1. The effects of circumferentially-applied stretch on the spontaneous contractility of a whole mount preparation of the guinea-pig upper urinary tract (UUT) (renal pelvis and ureter) were investigated by use of standard isometric tension recording techniques.
2. Simultaneous tension recordings of the proximal and distal portions of the renal pelvis (RP) and ureter revealed that spontaneous contractions, in 79% (n=66) of preparations, originated in the proximal RP (at a frequency of 4.5 min⁻¹) and propagated to the distal RP and ureter at a velocity of 1–3 cm s⁻¹. Pretreatment with tetrodotoxin (TTX) (3–10 μM) or N^G-nitro-L-arginine (100 μM) had little effect on the spontaneous contractility of the UUT, motility indexes (MIs) (contraction amplitude×contraction frequency) calculated after 20 min exposure were little affected by TTX or N^G-nitro-L-arginine (L-NOARG). ω-Conotoxin GVIA (100 nM) significantly reduced MI values in both the proximal RP and ureter.
3. Exposure of the spontaneously-active UUT to capsaicin (10 μM for 15 min) induced a transient increase in UUT contractility, followed by a prolonged negative inotropic effect. The MI values, calculated 60 min after the washout of capsaicin, for the proximal and distal RP and ureter were reduced to 56%, 53% (n=18) and 61% (n=16), respectively, of their control values. This capsaicin pretreatment blocked the positive inotropic effects of transmural electrical nerve stimulation on UUT contractility to reveal a small inhibitory effect which was readily blocked by tetrodotoxin (3 μM) (n=3). The excitatory and inhibitory actions of nerve stimulation were both blocked by TTX (3 μM)
4. A second exposure to capsaicin (10 μM for 15 min), further reduced the MI values (calculated 60 min after washout) in the proximal and distal RP to 41% and 31%, respectively (n=6; P<0.05), of the initial control values.
5. In 61% (n=99) of preparations, the application of stretch to the proximal RP (0.5 to 2 mm) evoked a decrease in the amplitude of the contractions recorded in the distal RP, but not in the ureter. Stretch applied to the distal RP or ureter had no effect on the contractions recorded in the other regions of the UUT.
6. In 5 out of 6 preparations, a single application of capsaicin (10 μM for 15 min) had little effect on the change in contractile force of the distal RP evoked upon stretch of the proximal RP.
7. The inhibition of the distal RP upon stretch of the proximal RP was partially reduced (P<0.05) when the UUT was pretreated with the calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP (8–37) (1 μM).
8. The application of the CGRP receptor agonist, hCGRP (100 nM) inhibited contractility in the UUT in a region dependent manner. The MI of the proximal RP was decreased 32% after 6 min; while the MIs of the distal RP and ureter were reduced 83% and 63%, respectively, within 5 min of the application of hCGRP.
9. Glibenclamide (1 μM) had little effect on the spontaneous contractility of the UUT, but significantly reduced the inhibition of the distal RP evoked upon stretch (0.5 to 2 mm) of the proximal RP. TTX (3–10 μM), L-NOARG (100 μM) or ω-conotoxin GVIA (100 nM) had little effect on the stretch-evoked inhibition of the distal RP.
10. It was concluded that circumferential stretch of the proximal RP inhibits the contractility of the distal RP and that a component of this inhibition involves the activation of a glibenclamide-sensitive mechanism via the release of endogenous CGRP, possibly from the varicosities of intramural sensory nerves.

     

Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery

1. In the rat hepatic artery, the acetylcholine-induced relaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) is abolished by a combination of apamin and charybdotoxin, inhibitors of small (SK_Ca) and large (BK_Ca) conductance calcium-sensitive potassium (K)-channels, respectively, but not by each toxin alone. The selective BK_Ca inhibitor iberiotoxin cannot replace charybdotoxin in this combination. Since delayed rectifier K-channels (K_V) represent another target for charybdotoxin, we explored the possible involvement of K_V in EDHF-mediated relaxation in this artery.
2. The K_V inhibitors, agitoxin-2 (0.3 μM), kaliotoxin (0.3 μM), β-dendrotoxin (0.3 μM), dofetilide (1 μM) and terikalant (10 μM), each in combination with apamin (0.3 μM) had no effect on the EDHF-mediated relaxation induced by acetylcholine in the presence of N^ω-nitro-L-arginine (0.3 mM) and indomethacin (10 μM), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n=2–3). Although the K_V inhibitor margatoxin (0.3 μM) was also without effect (n=5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC₅₀ and E_max values were 7.5±0.0 and 95±1% in the absence and 7.0±0.1 and 81±6% in the presence of margatoxin plus apamin, respectively; n=6; P<0.05).
3. Ciclazindol (10 μM) partially inhibited the EDHF-mediated relaxation by shifting the acetylcholine-concentration-response curve 12 fold to the right (n=6; P<0.05) and abolished the response when combined with apamin (0.3 μM; n=6). This combination did not inhibit acetylcholine-induced relaxations mediated by endothelium-derived NO (n=5).
4. A 4-aminopyridine-sensitive delayed rectifier current (I_K(V)) was identified in freshly-isolated single smooth muscle cells from rat hepatic artery. None of the cells displayed a rapidly-activating and -inactivating A-type current. Neither charybdotoxin (0.3 μM; n=3) nor ciclazindol (10 μM; n=5), alone or in combination with apamin (0.3 μM; n=4–5), had an effect on I_K(V). A tenfold higher concentration of ciclazindol (0.1 mM, n=4) markedly inhibited I_K(V), but this effect was not increased in the additional presence of apamin (0.3 μM; n=2).
5. By use of membranes prepared from rat brain cortex, [¹²⁵I]-charybdotoxin binding was consistent with an interaction at a single site with a K_D of approximately 25 pM. [¹²⁵I]-charybdotoxin binding was unaffected by iberiotoxin (0.1 μM, n=6), but was increased by apamin in a concentration-dependent manner (E_max 43±10%, P<0.05 and pEC₅₀ 7.1±0.2; n=7–8). Agitoxin-2 (10 nM) displaced [¹²⁵I]-charybdotoxin binding by 91±3% (n=6) and prevented the effect of apamin (1 μM; n=6).
6. It is concluded that the EDHF-mediated relaxation in the rat hepatic artery is not mediated by the opening of either K_V or BK_Ca. Instead, the target K-channels for EDHF seem to be structurally related to both K_V and BK_Ca. The possibility that a subtype of SK_Ca may be the target for EDHF is discussed.