Tamoxifen dilates porcine coronary arteries: roles for nitric oxide and ouabain-sensitive mechanisms

BACKGROUND AND PURPOSE: Experiments were designed to determine the mechanism of the relaxation induced by tamoxifen in porcine coronary arteries at the tissue, cellular and molecular levels. EXPERIMENTAL APPROACH: Porcine left circumflex coronary arteries were isolated and isometric tension was measured. [Ca2+]i in native endothelial cells of intact arteries was determined by a calcium fluorescence imaging technique and eNOS ser1177 phosphorylation was assayed by Western blotting. KEY RESULTS: Tamoxifen induced an endothelium-dependent relaxation that was antagonized by ICI 182,780 and abolished by NG-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). L-Arginine reversed the effect of L-NAME while indomethacin was without effect. Tamoxifen-induced relaxation was attenuated by charybdotoxin (CTX) plus apamin, ouabain or by incubation in a K+ -free solution. Moreover, tamoxifen triggered extracellular Ca2+ -dependent increases in endothelial [Ca2+]i and this effect was abolished by ICI 182,780. Endothelium-independent relaxation to sodium nitroprusside was also inhibited by ouabain or in a K+ -free solution. Furthermore, tamoxifen increased endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177 and ICI 182,780 prevented this effect. CONCLUSIONS AND IMPLICATIONS: The present results suggest that tamoxifen mainly induces endothelium-dependent relaxation and that endothelial nitric oxide (NO) is the primary mediator of this effect. NO-dependent responses may result from elevated [Ca2+]i in endothelial cells; an effect abolished by ICI 182,780. NO activates Na+/K+ -ATPase in vascular smooth muscle, leading to relaxation. These results suggest that tamoxifen is able to modulate eNOS phosphorylation directly.     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Regulation of apical and basolateral K+ conductances in the rat colon

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K⁺ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K⁺ in the rat proximal and distal colon.
2. Administration of nystatin (100 μg ml⁻¹ at the mucosal side), in the presence of Na⁺ and in the presence of a serosally directed K⁺ gradient, stimulate a large increase in short-circuit current (I_SC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na⁺-K⁺-ATPase and of a current across a quinine-sensitive basolateral K⁺ conductance.
3. The pump current, measured as Na⁺-dependent or scilliroside-sensitive current in the absence of a K⁺ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5×10⁻⁶ mol l⁻¹).
4. The current across the basolateral K⁺ conductance, measured as current in the presence of a serosally directed K⁺ gradient either in the absence of Na⁺ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5×10⁻⁵ mol l⁻¹), but inhibited by forskolin (5×10⁻⁶ mol l⁻¹).
5. Basolateral K⁺ depolarization induced a negative I_SC in both colonic segments, which was inhibited by the K⁺ channel blocker quinine (10⁻³ mol l⁻¹ at the mucosal side), but was resistant to tetraethylammonium (5×10⁻³ mol l⁻¹ at the mucosal side). This K⁺ current across an apical K⁺ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl⁻ conductance under these conditions.
6. These results demonstrate that an increase in intracellular Ca²⁺ concentration induced by carbachol causes an increase in the basolateral and the apical K⁺ conductance, thereby inducing K⁺ secretion in parallel with an indirect support for Cl⁻ secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K⁺ conductance; a mechanism which might contribute to the inhibition of K⁺ absorption.

     

Signalling pathways in bradykinin- and nitric oxide-induced hypotension in the normotensive rat; role of K+-channels

1. Bradykinin and nitric oxide (NO) are potent hypotensive agents. In the present study, the role of K⁺-channels in the signalling pathways responsible for their hypotensive action was investigated in normotensive, anaesthetized rats. The rats were treated with ion-channel inhibitors before administration of bradykinin (2.8, 5.6, 28 and 56 nmol kg⁻¹, i.v.) followed in some of the protocols by nitroprusside (1.1, 3.5, 7, 14, and 28 nmol kg⁻¹, i.v.).
2. No attenuation of the hypotensive response to bradykinin was detected for inhibitors of the Na-K-Cl-cotransporter (30 μmol kg⁻¹ furosemide), the ATP-sensitive K⁺-channel (40 μmol kg⁻¹ glibenclamide), high conductance Ca²⁺-activated K⁺-channel (180 μmol kg⁻¹ tetraethylammonium, 54 μmol kg⁻¹ tetrabutylammonium, 35 nmol kg⁻¹ iberiotoxin, 35 nmol kg⁻¹ charybdotoxin) or the low conductance Ca²⁺-activated K⁺-channel (74 nmol kg⁻¹ apamin).
3. However, the voltage-sensitive K⁺-channel (I_A) inhibitor 4-aminopyridine (4.05–40.5 μmol kg⁻¹) induced a concentration-dependent (P<0.0001) attenuation of the hypotensive response (P<0.0001). Bradykinin had no effect on heart rate in anaesthetized rats and this observation was not altered by pretreatment with 4-aminopyridine.
4. 4-Aminopyridine (53 μmol kg⁻¹) also significantly attenuated the hypotensive response to nitroprusside (P<0.0003) without altering the heart rate concentration-response curve. Of the two Ca²⁺-activated K⁺-channel inhibitors tested on nitroprusside-induced hypotension, tetrabutylammonium induced a slight attenuation (P<0.0101), whereas iberiotoxin had no effect.
5. We therefore concluded that, although the acute hypotensive response to bradykinin in the normotensive rat is not mediated through nitric oxide synthesis, the hypotensive response to both agents was mediated through opening of voltage-sensitive K⁺-channels (I_A), resulting in a decrease in peripheral vascular resistance.

     

Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

BACKGROUND AND PURPOSE: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. EXPERIMENTAL APPROACH: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured. KEY RESULTS: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA. CONCLUSIONS AND IMPLICATIONS: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.     

SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil

1. The classical ATP sensitive K⁺ (K_ATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K⁺ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K⁺ channel openers, such as pinacidil and nicorandil.
2. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the K_ATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method.
3. In the cell-attached recordings (145 mM K⁺ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K⁺ channel in both the transfected cells.
4. In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR_2B/Kir6.2 and SUR_2A/Kir6.2 channels (EC₅₀ of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR_2B/Kir6.2 channel >100 times more potently than the SUR_2A/Kir6.2 (EC₅₀ of ∼10 μM and >500 μM, respectively).
5. Thus, nicorandil, but not pinacidil, preferentially activates the K_ATP channels containing SUR_2B. Because SUR_2A and SUR_2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR_2B may play a critical role in the action of nicorandil on the vascular type classical K_ATP channel.

     

Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells

1. The action of the anticonvulsant drug phenytoin on K⁺ currents was investigated in neuroblastoma cells by whole-cell voltage-clamp recording.
2. Neuroblastoma cells expressed an outward K⁺ current with a voltage- and time-dependence which resembled the delayed-rectifier K⁺ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n=65). Inhibition was concentration-dependent with a half-maximal inhibitory concentration of 30.9±0.8 μM.
3. The K⁺ current inhibition by phenytoin was voltage-dependent with block by phenytoin being relieved by depolarization.
4. The times taken to reach steady-state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K⁺ current nor the K⁺ channel availability were significantly altered by the blocking drug. A use-dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.
5. These results suggest that phenytoin affects K⁺ currents and that this effect might lead to a reduction in neuronal excitability.

     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed.
2. NO (10⁻⁷ M) induced only contraction while 10⁻⁶ M NO induced contraction followed by relaxation. Methylene blue (up to 10⁻⁴ M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10⁻⁶ M NO. Administration of 8-bromo-cyclic GMP (10⁻⁶–10⁻⁴ M) only induced relaxation.
3. Sodium nitroprusside (SNP; 10⁻⁷–10⁻⁵ M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10⁻⁸ M NO and 10⁻⁶ M SNP; it was increased by a factor of 2 during contraction with 10⁻⁷ M NO, and by a factor of 12 during relaxation with 3×10⁻⁶ M NO.
4. The NO-induced contractions were not affected by ryanodine (3×10⁻⁵ M) but were concentration-dependently reduced by nifedipine (10⁻⁸–10⁻⁷ M) and apamin (3×10⁻⁹–3×10⁻⁸ M).
5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca²⁺ influx through L-type Ca²⁺ channels, that might be activated in response to the closure of Ca²⁺-dependent K⁺ channels.

     

Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume,intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pH_i) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pH_i and migration of fMLP stimulated PMNs.
2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.
3. Inhibition of the Na⁺/H⁺ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.
4. Step omission of extracellular Na⁺ caused intracellular acidification, which was accelerated by subsequent addition of gastric H⁺/K⁺ ATPase inhibitor SCH 28080, or by omission of extracellular K⁺ ions. In addition Na⁺ removal caused cell swelling, which was further enhanced by fMLP.
5. H⁺/K⁺ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.
6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na⁺/H⁺ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.
7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.
8. Western analysis with a monoclonal anti gastric H⁺/K⁺ATPase β-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.
9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na⁺/H⁺ exchanger and a K⁺-dependent H⁺-extruding mechanism, presumably an H⁺/K⁺ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

     

Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1. We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol.
2. Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED_50(M) of 0.43±0.15 nmol and a maximum relaxation (R_max(M) of 89.6±1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED_50(F)=0.72±0.19 nmol and R_max(F)=90.7±0.9%; n=22).
3. In the males, the addition of 100 μM N^G-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED_50(M)=6.45±3.53 nmol) and significantly (P<0.01) reduced R_max(M) (79.7±2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED_50(F)=0.89±0.19 nmol, R_max(F)=86.9±2.3%, n=9).
4. Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (R_max(M)=40.3±9.2%, n=4; P<0.001) and female rats by 1.5 fold (R_max(F)=55.3±3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED_50(M)=0.87±0.26 nmol; P<0.01) but not the females (ED_50(F)=4.04±1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups.
5. The cannabinoid receptor antagonist SR141716A (1 μM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED_50(M)=1.41±0.74 nmol, R_max(M)=89.4±2.5%, n=7; ED_50(F)=2.17±0.95 nmol, R_max(F)=89.9±1.8%, n=9). In mesenteries from male rats, in the presence of 100 μM L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED_50(M)=53.8±36.8 nmol) without affecting R_max(M) (72.4±4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR141716A, significantly (P<0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED_50(F)=6.66±2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased R_max(F) (70.1±5.5%, n=8).
6. Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds.
7. The continuous presence of sodium nitroprusside (SNP; 20–60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED_50(M)=40.9±19.6 nmol, R_max(M)=79.4±2.5%, n=11). Similarly in mesenteries from female rats, the ED_50(F) was also significantly (P<0.01) increased 7 fold (6.24±2.02 nmol), while the R_max(F) was unaffected (81.9±11.0%; n=4).
8. The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males.

     

Endothelium-dependent relaxation by substance P in human isolated omental arteries and veins: relative contribution of prostanoids,nitric oxide and hyperpolarization

1. The objective of the present study was to investigate human omental arteries and veins with respect to: (i) the contractile effect of the thromboxane A₂ analogue U46619, (ii) endothelium-dependency and mediators of the relaxing effect of substance P (SP) and acetylcholine (ACh).
2. Changes in isometric tension in response to administration of U46619, SP and ACh were measured in human isolated omental arteries and veins with and without endothelium. To investigate the mechanism of action of SP, the SP-induced relaxation was measured in the presence of indomethacin (cyclo-oxygenase inhibitor), N^G-monomethyl-L-arginine (L-NMMA, nitric oxide-synthase inhibitor), KCl (inhibitor of endothelium-dependent hyperpolarization), tetraethylammonium (TEA; non-selective inhibitor of K⁺-channels, with some preference for the high conductance Ca²⁺-activated K⁺-channel, BK_Ca), glibenclamide (inhibitor of the ATP-sensitive K⁺-channel) and/or clotrimazole (inhibitor of the cytochrome P450-system and the intermediate conductance Ca²⁺-activated K⁺-channel, IK_Ca).
3. U46619 contracted both the artery and the vein segments. Endothelium removal did not alter the contraction.
4. ACh caused neither contraction nor relaxation in artery and vein segments precontracted with U46619.
5. In both artery and vein segments precontracted with U46619, SP produced endothelium-dependent relaxation. The relaxation was unaffected by indomethacin, but was incompletely reduced by L-NMMA and KCl respectively. The L-NMMA-resistent relaxation was abolished in the presence of KCl.
6. TEA inhibited the SP-induced relaxation in artery and vein segments both in the presence and absence of L-NMMA and indomethacin, while glibenclamide and clotrimazole had no effect.
7. In conclusion, the SP-induced relaxation in human omental arteries and veins seems to be mediated via NO and endothelium-dependent hyperpolarization. K_ATP and I_KCa are probably not involved in the hyperpolarization, but activation of BK_Ca may contribute to the hyperpolarization. Prostanoid synthesis and the cytochrome P450-system are probably not involved in the SP-induced relaxation in this area.

     

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes

1. The effects on the voltage-gated (I_K) and Ca²⁺ activated (I_K,Ca) K⁺ currents in rat arterial myocytes of the 5-lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated.
2. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration-dependent reduction of the I_K, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886.
3. MK886 application led to a concentration-dependent increase in the amplitude of the TEA-sensitive I_K,Ca current and single channel activity in RPAMs in whole cell and inside-out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4–5 fold increase was observed at 10 μM MK886. MK886 also increased I_K,Ca in rat mesenteric arterial myocytes (RMAMs).
4. L538,916, an analogue of MK886 which does not block FLAP, had no effect on either I_K or I_K,Ca at a concentration of 10 μM.
5. Leukotriene C₄ (100 nM) had no effect on either I_K or I_K,Ca in RPAMs. MK886 produced its usual increase in I_K,Ca and also blocked I_K, in the presence of leukotriene C₄. Similarly, leukotriene E₄ (100 nM) did not alter the amplitude of I_K. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect I_K in RPAMs, and did not affect the response to MK886.
6. Arachidonic acid (10 μM) enhanced I_K,Ca in both RPAMs and RMAMs.
7. The results show that MK886 markedly affects both I_K and I_K,Ca in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K⁺ channels, and mimic its effects. Alternatively, MK886 might act via non-selective effects on other arachidonic acid metabolites which could modify K⁺ channel function.

     

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K⁺ channel (hKv1.5) stably expressed in Ltk⁻ cells and using the whole-cell configuration of the patch-clamp technique.
2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K_D values of 4.4±0.3 μM and 9.2±1.6 μM, respectively.
3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17±0.55 (n=10) and 0.16±0.81 (n=16).
4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9±0.9)×10⁶ M⁻¹ s⁻¹ and 39.5±4.2 s⁻¹, respectively. For 5-hydroxy-propafenone these values averaged (2.3±0.3)×10⁶ M⁻¹ s⁻¹ and 21.4±3.1 s⁻¹, respectively.
5. Both drugs reduced the tail current amplitude recorded at −40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a `crossover'' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed.
6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied.
7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

     

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg

1. The sea urchin egg homogenate is an ideal model to characterize Ca²⁺-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP₃- and ryanodine-sensitive Ca²⁺-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.
2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca²⁺-releasing agent, NAADP, compared to inositol trisphosphate (InsP₃) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.
3. NAADP-induced Ca²⁺-release was blocked by L-type Ca²⁺-channel blockers and by Bay K 8644, while InsP₃- and cyclic ADPR-induced Ca²⁺-release were insensitive to these agents. L-type Ca²⁺-channel blockers did not displace [³²P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion.
4. Similarly, a number of K⁺-channel antagonists blocked NAADP-induced Ca²⁺-release selectively over InsP₃- and cyclic ADPR-induced Ca²⁺-release. Radioligand studies showed that these agents were not competitive antagonists.
5. As has been shown for InsP₃ and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca²⁺-release mechanisms.
6. The presence of K⁺ was not essential for NAADP-induced Ca²⁺-release, since substitution of K⁺ with other monovalent cations in the experimental media did not significantly alter Ca²⁺ release by NAADP. On the contrary, cyclic ADPR and InsP₃-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K⁺. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca²⁺-release. While the alkaline condition permitted InsP₃ and cyclic ADPR-induced Ca²⁺-release, the acidic condition completely hampered both Ca²⁺-release mechanisms.
7. The present results characterize pharmacologically and biochemically the novel Ca²⁺-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

     

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.

     

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.

     

Role of neuronal voltage-gated K(+) channels in the modulation of the nitrergic neurotransmission of the pig urinary bladder neck

BACKGROUND AND PURPOSE: As nitric oxide (NO) plays an essential role in the inhibitory neurotransmission of the bladder neck of several species, the current study investigates the mechanisms underlying the NO-induced relaxations in the pig urinary bladder neck. EXPERIMENTAL APPROACH: Urothelium-denuded bladder neck strips were dissected and mounted in isolated organ baths containing a physiological saline solution at 37 degrees C and continuously gassed with 5% CO(2) and 95% O(2), for isometric force recording. The relaxations to transmural nerve stimulation (EFS), or to exogenously applied acidified NaNO(2) solution were carried out on strips pre-contracted with phenylephrine, and treated with guanethidine and atropine, to block noradrenergic neurotransmission and muscarinic receptors, respectively. KEY RESULTS: EFS (0.2-1 Hz) and addition of acidified NaNO(2) solution (1 microM-1 mM) evoked frequency- and concentration-dependent relaxations, respectively. These responses were potently reduced by the blockade of guanylate cyclase and were not modified by the K(+) channel blockers iberiotoxin, charybdotoxin, apamin or glibenclamide. The voltage-gated K(+) (Kv) channels inhibitor 4-aminopyridine, greatly enhanced the nitrergic relaxations evoked by EFS, but did not affect the NaNO(2) solution-induced relaxations. CONCLUSIONS AND IMPLICATIONS: NO, whose release is modulated by pre-junctional Kv channels, relaxes the pig urinary bladder neck through a mechanism dependent on the activation of guanylate cyclase, in which post-junctional K(+) channels do not seem to be involved. Modulation of Kv channels could be useful in the therapy of the urinary incontinence produced by intrinsic sphincteric deficiency.     

A discriminant block among K+ channel types by phenytoin in neuroblastoma cells

1. The action of the anticonvulsant drug phenytoin on K⁺ channels was investigated in neuroblastoma cells (N2A) by using the single-channel patch-clamp technique.
2. N2A cells expressed three types of delayed rectifier K⁺ channels, which were found to have a conductance of 10–20 pS in a `physiological'' K⁺ gradient. When added to the external solution at concentrations ranging between 1 and 200 μM, phenytoin decreased single channel activity, whereas the unitary current amplitude was unaffected in all three types of channels.
3. The open probability of the biggest channel decreased, according to an exponential distribution of open and closed times, from 40% in control conditions to 10% in the presence of 50 μM phenytoin (Vm=40 mv). The reduction in the open-channel probability was concentration-dependent with a IC₅₀=27.2±0.9 μM.
4. A transient type of K⁺ channel was identified that was affected by cumulative inactivation and had a conductance of a mean value equal to 26 pS. Finally, a voltage-and Ca²⁺-dependent K⁺ channel with a unitary conductance of 95 pS was recorded. Both the channel''s amplitude and kinetics were unaffected by phenytoin.
5. These results confirm the phenytoin effect on K⁺ currents and suggest that the drug may be considered a selective blocker of delayed rectifier K⁺ channels.

     

Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors

1. PD 81,723 has been shown to enhance binding of adenosine to A₁ receptors by stabilizing G protein-receptor coupling (‘allosteric enhancement''). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism.
2. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K⁺ current (I_K(ACh)) activated by different G_i-coupled receptors (A₁, M₂, sphingolipid). PD 81,273 caused inhibition of I_K(ACh) (IC₅₀≃5 μM) activated by either of the three receptors. Receptor-independent I_K(ACh) in cells loaded with GTP-γ-S and background I_K(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected.
3. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 μM) via the patch pipette did not affect either I_K(ACh) or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K⁺ channels; this is supported by the finding that ventricular I_K1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming I_K(ACh) but is not G protein-gated, was also blocked by this compound.
4. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A₁ adenosine receptor-G_i-I_K(ACh) pathway.