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.

     

Neuroregulation of mucus secretion by opioid receptors and KATP and BKCa channels in ferret trachea in vitro

1. Opioid agonists inhibit neurogenic mucus secretion in the airways. The mechanism of the inhibition is unknown but may be via opening of potassium (K⁺) channels. We studied the effect on neurogenic secretion in ferret trachea in vitro of the OP₁ receptor (formerly known as δ opioid receptor) agonist [D-Pen^2,5]enkephalin (DPDPE), the OP₂ receptor (formely κ) agonist U-50,488H, the OP₃ receptor (formerly μ) agonist [D-Ala², N-Me-Phe, Gly-ol⁵]enkephalin (DAMGO), the ATP-sensitive K⁺ (K_ATP) channel inhibitor glibenclamide, the large conductance calcium activated K⁺ (BK_Ca) channel blocker iberiotoxin, the small conductance K_Ca (SK_Ca) channel blocker apamin, the K_ATP channel opener levcromakalim, a putative K_ATP channel opener RS 91309, and the BK_Ca channel opener NS 1619. Secretion was quantified by use of ³⁵SO₄ as a mucus marker.
2. Electrical stimulation increased tracheal secretion by up to 40 fold above sham-stimulated levels. DAMGO or DPDPE (10 μM each) significantly inhibited neurogenic secretion by 85% and 77%, respectively, effects which were reversed by naloxone. U-50,488H had no significant inhibitory effect on neurogenic secretion, and none of the opioids had any effect on ACh-induced or [Sar⁹]substance P-induced secretion.
3. Inhibition of neurogenic secretion by DAMGO or DPDPE was reversed by iberiotoxin (3 μM) but not by either glibenclamide or apamin (0.1 μM each). Iberiotoxin alone did not affect the neurogenic secretory response.
4. Levcromakalim, RS 91309 or NS 1619 (3 nM–3 μM) inhibited neurogenic secretion with maximal inhibitions at 3 μM of 68%, 72% and 96%, respectively. Neither levcromakalim nor RS 91309 at any concentration tested significantly inhibited acetylcholine (ACh)-induced secretion, whereas inhibition (60%) was achieved at the highest concentration of NS 1619, a response which was blocked by iberiotoxin.
5. Inhibition of neurogenic secretion by levcromakalim (3 μM) or RS 91309 (30 nM) was inhibited by glibenclamide but not by iberiotoxin. In contrast, inhibition by NS 1619 (30 nM and 3 μM) was blocked by iberiotoxin but not by glibenclamide.
6. We conclude that, in ferret trachea in vitro, OP₁ or OP₃ opioid receptors inhibit neurogenic mucus secretion at a prejunctional site and that the mechanism of the inhibition is via opening of BK_Ca channels. Direct opening of BK_Ca channels or K_ATP channels also inhibits neurogenic mucus secretion. In addition, opening of BK_Ca channels inhibits ACh-evoked secretion of mucus. Drugs which open BK_Ca channels may have therapeutic anti-secretory activity in bronchial diseases in which neurogenic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

     

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.

     

Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K+ channels in pig urethral myocytes

1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches).
2. Levcromakalim produced a concentration-dependent outward current at a holding potential of −50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations.
3. In conventional whole-cell configuration, the levcromakalim (100 μM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min.
4. The peak amplitude of the levcromakalim (100 μM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide.
5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 μM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used.
6. In the cell-attached mode in symmetrical 140 mM K⁺ conditions, glibenclamide inhibited the 100 μM levcromakalim-activated 43 pS K⁺ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (K_i) of approximately 520 nM.
7. In inside-out patches in which the glibenclamide-sensitive K⁺ channel had run down after exposure to levcromakalim, both uridine 5′-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg²⁺ to the UDP-reactivated K⁺ channels enhanced the channel activity reversibly.
8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K⁺ channel without levcromakalim, providing that there was free Mg²⁺ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg²⁺). Additional application of levcromakalim caused a further reversible activation of channel opening.
9. In the presence of levcromakalim, application of adenosine 5′-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner.
10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K⁺ channel in the presence of 100 μM levcromakalim in inside-out patches.
11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K⁺ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg²⁺ may play an important role in regulating the channel activity.

     

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.

     

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.

     

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.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

Effects of mexiletine on ATP sensitive K+ channel of rat skeletal muscle fibres: a state dependent mechanism of action

1. The effects of mexiletine were evaluated on the ATP-sensitive K⁺ channel (K_ATP) of rat skeletal muscle fibres using patch clamp techniques. The effects of mexiletine were studied on macropatch currents 20 s (maximally activated), 8 min (early stage of rundown) and 15 min (late stage of rundown) after excision in the absence or in the presence of internal ADP (50–100 μM) or UDP (500 μM). In addition, the effects of mexiletine were tested on single channel.
2. In the absence of ADP and UDP, mexiletine inhibited the current through maximally activated channels with an IC₅₀ of −5.58±0.3 M. Nucleoside diphosphates shifted the current versus mexiletine concentration relationship to the right on the log concentration axis. UDP (500 μM) was more efficacious than ADP (50–100 μM) in this effect.
3. At the early stage of rundown, the sensitivity of the channel to mexiletine was reduced and nucleoside diphosphates, particularly UDP, antagonized the effect of mexiletine. At the late stage of rundown, mexiletine did not affect the currents.
4. At the single channel level, 1 μM mexiletine reduced the mean burst duration by 63% and prolonged the arithmetic mean closed time intervals between the bursts of openings without altering the open time and closed time distributions. Mexiletine did not affect the single channel conductance.
5. These results show that in skeletal muscle, mexiletine is a state-dependent K_ATP channel inhibitor which either acts through the nucleotide binding site or a site allosterically coupled to it.

     

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.

     

Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of β-cells

1. The structure activity relationships for the insulin secretagogues N-benzoyl-D-phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT-T15 and mouse pancreatic β-cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for K_ATP-channel inhibition. In addition, the effects of cytosolic nucleotides on K_ATP-channel inhibition by NBDP were investigated.
2. NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (K_D value) of 11 μM and half-maximally effective concentrations of K_ATP-channel inhibition (EC₅₀ values) between 2 and 4 μM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP).
3. In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1–1 mM) reduced K_ATP-channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), K_ATP-channel activity was completely suppressed by 0.1 mM NBDP.
4. The L-isomer of N-benzoyl-phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D-isomer.
5. Introduction of a p-nitro substituent in the D-phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold.
6. Introduction of a p-amino substituent in the D-phenylalanine moiety of NBDP (N-benzoyl-p-amino-D-phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative.
7. Replacing the benzene ring in the D-phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the K_D and EC₅₀ values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency.
8. This study shows that N-acylphenylalanines interact with the sulphonylurea receptor of pancreatic β-cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N-acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of K_ATP-channel activity in the absence of inhibitory cytosolic nucleotides.

     

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.

     

Modulation of relaxation to levcromakalim by S-nitroso-N-acetylpenicillamine (SNAP) and 8-bromo cyclic GMP in the rat isolated mesenteric artery

1. Levcromakalim caused concentration-dependent relaxations of methoxamine-induced tone in both endothelium-denuded and intact vessels. Its potency was reduced by the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP; 0.1 μM or 1 μM) in both denuded and intact vessels. The maximal relaxation (R_max) was reduced only in denuded vessels.
2. SNAP was more potent in endothelium-denuded than intact vessels but there were no differences in R_max. Glibenclamide (10 μM) did not affect relaxation to SNAP in endothelium-denuded or intact vessels.
3. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) increased the potency and R_max of levcromakalim in endothelium-intact vessels. ODQ had no effect in denuded vessels.
4. ODQ (10 μM) reduced the vasorelaxant potency of SNAP in both intact and endothelium-denuded vessels by 190-fold and 620-fold, respectively.
5. 8-bromo cyclic GMP (10 or 30 μM) reduced both the potency and R_max of levcromakalim in de-endothelialized vessels, but had no effect in intact vessels although it reduced both the potency and R_max of levcromakalim in intact vessels incubated with ODQ (10 μM).
6. In the presence of ODQ (10 μM), SNAP (0.1 μM or 1 μM) reduced the potency of levcromakalim in intact vessels, without altering R_max, but had no effect in denuded vessels. SNAP (50 μM) reduced both the potency and R_max of levcromakalim in intact and endothelium-denuded vessels.
7. Therefore, although SNAP causes relaxation principally through generation of cyclic GMP, it can modulate the actions of levcromakalim through mechanisms both dependent on, and independent of, cyclic GMP; the former predominate in endothelium-denuded vessels and the latter in intact vessels.

     

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.

     

Resistance to myocardial infarction induced by heat stress and the effect of ATP-sensitive potassium channel blockade in the rat isolated heart

1. Heat stress (HS) is known to protect against myocardial ischaemia-reperfusion injury by improving mechanical dysfunction and decreasing necrosis. However, the mechanisms responsible for this form of cardioprotection remain to be elucidated. ATP-sensitive potassium (K_ATP) channels have been shown to be involved in the delayed phase of protection following ischaemic preconditioning, a phenomenon closely resembling the HS-induced cardioprotection. The aim of this study was thus to investigate the role of K_ATP channels in HS-induced protection of the isolated rat heart.
2. Twenty four hours after whole body heat stress (at 42°C for 15 min) or sham anaesthesia, isolated perfused hearts were subjected to a 15 min stabilization period followed by a 15 min infusion of either 10 μM glibenclamide (Glib), 100 μM sodium 5-hydroxydecanoate (5HD) or vehicle (0.04% DMSO). Regional ischaemia (35 min) and reperfusion (120 min) were then performed.
3. Prior heat stress significantly reduced infarct-to-risk ratio (from 42.4±2.4% to 19.4±2.9, P<0.001). This resistance to myocardial infarction was abolished in both Glib-treated (40.1±1.8% vs 42.3±1.8%) and 5HD-treated (41.2±1.8% vs 41.8±1.2%) groups.
4. The results of this study suggest that K_ATP channel activation contributes to the cytoprotective response induced by heat stress.

     

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.

     

Involvement of a glibenclamide-sensitive mechanism in the nitrergic neurotransmission of the pig intravesical ureter

1. The present study was designed to investigate whether potassium (K⁺) channels are involved in the relaxations to nitric oxide (NO) of pig intravesical ureteral preparations suspended in organ baths for isometric tension recordings. In ureteral strips treated with guanethidine (10⁻⁵ M) and atropine (10⁻⁷ M) to block adrenergic neurotransmission and muscarinic receptors, respectively, NO was either released from nitrergic nerves by electrical field stimulation (EFS, 0.5–10 Hz, 1 ms duration, 20 s trains), or exogenously-applied as an acidified solution of sodium nitrite (NaNO₂, 10⁻⁶–10⁻³ M).
2. Incubation with an inhibitor of guanylate cyclase activation by NO, methylene blue (10⁻⁵ M) did not change the basal tension of intravesical ureteral strips but inhibited the relaxation induced by EFS or exogenous NO on ureteral preparations contracted with the thromboxane analogue U46619 (10⁻⁷ M).
3. Incubation with charybdotoxin (3×10⁻⁸ M) and apamin (5×10⁻⁷ M), which are inhibitors of large and small conductance calcium (Ca²⁺)-activated K⁺ channels, respectively, did not modify basal tension or the relaxations induced by EFS and exogenous NO. Treatment with charybdotoxin or apamin plus methylene blue (10⁻⁵ M) significantly reduced the relaxations to EFS and exogenous NO. However, in both cases the reductions were similar to the inhibition evoked by methylene blue alone. The combined addition of charybdotoxin plus apamin did not change the relaxations to EFS or exogenously added NO of the porcine intravesical ureter.
4. Cromakalim (10⁻⁸–3×10⁻⁶ M), an opener of ATP-sensitive K⁺ channels, evoked a dose-dependent relaxation with a pD₂ of 7.3±0.2 and maximum relaxant effect of a 71.8±4.2% of the contraction induced by U46619 in the pig intravesical ureter. The blocker of ATP-sensitive K⁺ channels, glibenclamide (10⁻⁶ M), inhibited markedly the relaxations to cromakalim.
5. Glibenclamide (10⁻⁶ M) had no effect on the basal tone of ureteral preparations but significantly reduced the relaxations induced by both EFS and exogenous NO. Combined treatment with methylene blue (10⁻⁵ M) and glibenclamide (10⁻⁶ M) did not exert an effect greater than that of methylene blue alone on either EFS- or NO-evoked relaxations of the pig ureter.
6. The present results suggest that NO acts as an inhibitory neurotransmitter in the pig intravesical ureter and relaxes smooth muscle through a guanylate cyclase-dependent mechanism which seems to favour the opening of glibenclamide-sensitive K⁺ channels.

     

The effect of glibenclamide on the production of interstitial adenosine by inhibiting ecto-5′-nucleotidase in rat hearts

1. Adenosine exerts cardioprotective effects on the ischaemic myocardium. The production of adenosine in the ischaemic myocardium is attributed primarily to the enzymatic dephosphorylation of adenosine 5′-monophosphate (AMP) by 5′-nucleotidase. We determined the activity of 5′-nucleotidase in rat hearts. The objective of the study was to determine the effects of ATP-sensitive K⁺ (K_ATP) channel antagonists (glibenclamide and 5-hydroxydecanoate) on the production of adenosine, by use of a flexibly mounted microdialysis technique.
2. Rats were anaesthetized and the microdialysis probe was implanted in the left ventricular myocardium, followed by perfusion with Tyrode solution. The baseline level of dialysate adenosine was 0.51±0.09 μM (n=16). Introduction of AMP (100 μM) through the probe increased the dialysate adenosine markedly to 9.79±0.43 μM (n=12, P<0.001 vs baseline), and this increase was inhibited by the ecto-5′-nucleotidase inhibitor, α,β-methyleneadenosine 5′-diphosphate (100 μM), to 0.76±0.12 μM (n=8). Thus, the dialysate adenosine noted during the perfusion of AMP originated from dephosphorylation of AMP by ecto-5′-nucleotidase, and the dialysate level of adenosine attained reflects the ecto-5′-nucleotidase activity in the tissue in situ.
3. Glibenclamide (0.1–100 μM) decreased the adenosine concentration measured during the perfusion of AMP (100 μM) in a concentration-dependent manner (IC₅₀=10.5 μM). In contrast, 5-hydroxydecanoate (10–100 μM) did not affect the concentrations of dialysate adenosine, measured in the presence of AMP (100 μM). These results suggest that glibenclamide inhibits the activity of endogenous ecto-5′-nucleotidase and decreases the concentration of adenosine in the interstitial space of rat ventricular muscles in situ.

     

The mechanisms of enhancement and inhibition of field stimulation responses of guinea-pig vas deferens by prostacyclin analogues

1. In the guinea-pig isolated vas deferens preparation bathed in Tyrode''s solution, the prostacyclin analogues, cicaprost, TEI-9063, iloprost, taprostene and benzodioxane-prostacyclin, enhanced twitch responses to submaximal electrical field stimulation (20%-EFS). The high potency of cicaprost (EC₁₅₀=1.3 nM) and the relative potencies of the analogues (equi-effective molar ratios=1.0, 0.85, 1.6, 17 and 82, respectively) suggest the involvement of a prostacyclin (IP-) receptor.
2. Maximum enhancement induced by cicaprost in 2.5 mM K⁺ Krebs-Henseleit solution was similar to that in Tyrode solution (2.7 mM K⁺), but was progressively reduced as the K⁺ concentration was increased to 3.9, 5.9 and 11.9 mM. There was also a greater tendency for the other prostacyclin analogues to inhibit EFS responses in 5.9 mM standard K⁺ Krebs-Henseleit solution; this may be attributed to their agonist actions on presynaptic EP₃-receptors resulting in inhibition of transmitter release.
3. The EFS enhancing action of cicaprost was not affected by the α₁-adrenoceptor antagonist prazosin (100 and 1000 nM). Cicaprost (20 and 200 nM) did not affect contractile responses of the vas deferens to either ATP (5 μM) or α,β-methylene ATP (1 μM) in the presence of tetrodotoxin (TTX, 100 nM). In addition, enhancement by cicaprost of responses to higher concentrations of ATP (30 and 300 μM) in the absence of TTX, as shown previously by others, was not seen. Prostaglandin E₂ (PGE₂, 10 nM) and another prostacyclin analogue TEI-3356 (20 nM) enhanced purinoceptor agonist responses. Unexpectedly, TTX (0.1 and 1 μM) partially inhibited contractions elicited by 10–1000 μM ATP; contractions elicited by 1–3 μM ATP were unaffected. Further studies are required to establish whether a pre- or post-synaptic mechanism is involved.
4. In a separate series of experiments, cicaprost (5–250 nM), TEI-9063 (3–300 nM), 4-aminopyridine (10–100 μM) and tetraethylammonium (100–1000 μM) enhanced both 20%-EFS responses and the accompanying overflow of noradrenaline to a similar extent. In further experiments with the EP₁-receptor antagonist AH 6809, TEI-3356 (1.0–100 nM) and the EP₃-receptor agonist, sulprostone (0.1–1.0 nM) inhibited both maximal EFS responses and noradrenaline overflow, thus confirming previous reports of the high activity of TEI-3356 at the EP₃-receptor. Cicaprost had no significant effect on noradrenaline overflow at 10 and 100 nM, but produced a modest inhibition at 640 nM.
5. In conclusion, our studies show that prostacyclin analogues (particularly TEI-3356) can inhibit EFS responses of the guinea-pig vas deferens by acting as agonists at presynaptic EP₃-receptors. Prostacyclin analogues (particularly cicaprost and TEI-9063) can also enhance EFS responses through activation of IP-receptors. The mechanism of the enhancement has not been rigorously established but from our results we favour a presynaptic action to increase transmitter release.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.