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

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

     

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery

1. The aim of the present study was to identify the sources of Ca²⁺ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release.
2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 μM indomethacin, 300 μM N^G-nitro-L-arginine or 10 μM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF.
3. In the presence of extracellular Ca²⁺, 1 μM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca²⁺-free medium, ACh produced only transient hyperpolarization.
4. Pretreatment with 100 nM thapsigargin and 3 μM cyclopiazonic acid, endoplasmic reticulum Ca²⁺-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 μM ryanodine.
5. In the presence of 5 mM Ni²⁺ or 3 mM Mn²⁺, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 μM nifedipine had no effect on ACh-induced hyperpolarization.
6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 μM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization.
7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca²⁺ release from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca²⁺ influx via a Ni²⁺- and Mn²⁺-sensitive pathway distinct from L-type Ca²⁺ channels. The Ca²⁺-influx mechanism seems to be activated following IP₃-induced depletion of the pool.

     

Hydrogen peroxide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

This study was designed to determine whether or not endothelium-dependent hyperpolarizations evoked by acetylcholine in the isolated guinea-pig carotid artery involve hydrogen peroxide. Membrane potential was recorded in the vascular smooth muscle cells of that artery. Under control conditions, acetylcholine induced endothelium-dependent hyperpolarization of the vascular smooth muscle cells which was not affected by the presence of catalase, superoxide dismutase or their combination. Neither the superoxide dismutase mimetic, tiron nor the thiol-reducing agent N-acetyl-l-cysteine modified the hyperpolarization evoked by 0.1 μM acetylcholine but each produced a partial and significant inhibition of the hyperpolarization induced by 1 μM acetylcholine. Neither 10 nor 100 μM hydrogen peroxide influenced the resting membrane potential of the smooth muscle cells and the higher concentration did not significantly influence the hyperpolarization elicited by acetylcholine. These data indicate that, in the guinea-pig isolated carotid artery, hydrogen peroxide is unlikely to contribute to the endothelium-dependent hyperpolarization evoked by acetylcholine.     

哇巴因和高钾对乙酰胆碱致家兔肺动脉内皮依赖性超极化的作用

采用标准微电极技术观察了哇巴因和高钾对乙酰胆碱（ａｃｅｔｙｌｃｈｏｌｉｎｅ，Ａｃｈ）致家兔肺动脉内皮依赖性超极化的作用。结果；Ａｃｈ１０μｍｏｌ·Ｌ－１可使内皮完整的肺动脉膜电位产生明显的超极化。该作用在去除内皮或预先用阿托品阻断Ａｃｈ受体的条件下消失。哇巴因可抑制无钾／复钾所致的膜电位超极化，但不影响Ａｃｈ所致的内皮依赖性超极化。高钾则可取消Ａｃｈ的内皮依赖性超极化。提示：Ａｃｈ引起的家兔肺动脉内皮依赖性超极化可能由细胞膜上钾通道开放所致，而和Ｎａ／Ｋ泵无关。     

Characteristics of ACh-induced hyperpolarization and relaxation in rabbit jugular vein

BACKGROUND AND PURPOSE The roles played by endothelium-derived NO and prostacyclin and by endothelial cell hyperpolarization in ACh-induced relaxation have been well characterized in arteries. However, the mechanisms underlying ACh-induced relaxation in veins remain to be fully clarified. EXPERIMENTAL APPROACH ACh-induced smooth muscle cell (SMC) hyperpolarization and relaxation were measured in endothelium-intact and -denuded preparations of rabbit jugular vein. KEY RESULTS In endothelium-intact preparations, ACh (≤10(-8) M) marginally increased the intracellular concentration of Ca(2+) ([Ca(2+) ](i) ) in endothelial cells but did not alter the SMC membrane potential. However, ACh (10(-10) -10(-8) M) induced a concentration-dependent relaxation during the contraction induced by PGF(2α) and this relaxation was blocked by the NO synthase inhibitor N(ω) -nitro-l-arginine. ACh (10(-8) -10(-6) M) concentration-dependently increased endothelial [Ca(2+) ](i) and induced SMC hyperpolarization and relaxation. These SMC responses were blocked in the combined presence of apamin [blocker of small-conductance Ca(2+) -activated K(+) (SK(Ca) , K(Ca) 2.3) channel], TRAM 34 [blocker of intermediate-conductance Ca(2+) -activated K(+) (IK(Ca) , K(Ca) 3.1) channel] and margatoxin [blocker of subfamily of voltage-gated K(+) (K(V) ) channel, K(V) 1]. CONCLUSIONS AND IMPLICATIONS In rabbit jugular vein, NO plays a primary role in endothelium-dependent relaxation at very low concentrations of ACh (10(-10) -10(-8) M). At higher concentrations, ACh (10(-8) -3 × 10(-6) M) induces SMC hyperpolarization through activation of endothelial IK(Ca) , K(V) 1 and (possibly) SK(Ca) channels and produces relaxation. These results imply that ACh regulates rabbit jugular vein tonus through activation of two endothelium-dependent regulatory mechanisms.     

C-type natriuretic peptide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

BACKGROUND AND PURPOSE: C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. EXPERIMENTAL APPROACH: The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. KEY RESULTS: Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of N (G)-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. CONCLUSIONS AND IMPLICATIONS: In the isolated carotid artery of the guinea-pig, CNP activates K(ATP) and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely.     

Openers of calcium-activated potassium channels and endothelium-dependent hyperpolarizations in the guinea pig carotid artery

This study was designed to determine whether putative openers of calcium-activated potassium channels of small and/or intermediate conductance (SK_Ca and IK_Ca) induce vascular smooth muscle hyperpolarizations and to identify the underlying mechanisms. The membrane potential of guinea pig carotid artery smooth muscle cells was recorded with intracellular microelectrodes in the presence of N ^ω-nitro-l-arginine and indomethacin. Acetylcholine and NS-309 produced endothelium-dependent hyperpolarizations. The effects of acetylcholine were partially and significantly inhibited by apamin. The combinations of charybdotoxin plus apamin and TRAM-34 plus apamin markedly and significantly reduced these hyperpolarizations. 1-ethyl-2-benzimidazolinone (1-EBIO) induced hyperpolarizations that were unaffected by TRAM-34 but partially inhibited by charybdotoxin, apamin, TRAM-34 plus apamin, and charybdotoxin plus apamin. Riluzole produced only marginal hyperpolarizations. Therefore, in the guinea pig carotid artery, endothelium-dependent hyperpolarization to acetylcholine involves the activation of both SK_Ca and IK_Ca, with a predominant role for the former channel. 1-EBIO is a non-selective and weak opener of SK_Ca, while riluzole is virtually ineffective. By contrast, NS-309 is a reasonably potent and selective opener of both SK_Ca and IK_Ca, and this compound mimics the endothelium-dependent hyperpolarizations to acetylcholine.     

Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

The three subtypes of calcium-activated potassium channels (K_Ca) of large, intermediate and small conductance (BK_Ca, IK_Ca and SK_Ca) are present in the vascular wall. In healthy arteries, BK_Ca channels are preferentially expressed in vascular smooth muscle cells, while IK_Ca and SK_Ca are preferentially located in endothelial cells. The activation of endothelial IK_Ca and SK_Ca contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na⁺/K⁺-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H₂O₂) hyperpolarize and relax the underlying smooth muscle cells by activating BK_Ca. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BK_Ca. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle K_Ca could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IK_Ca may prevent restenosis and that of BK_Ca channels sepsis-dependent hypotension.     

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.

     

Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease

1. The elusive nature of endothelium-derived hyperpolarizing factor (EDHF) has hampered detailed study of the ionic mechanisms that underlie the EDHF hyperpolarization and relaxation. Most studies have relied on a pharmacological approach in which interpretations of results can be confounded by limited specificity of action of the drugs used. Nevertheless, small-, intermediate- and large-conductance Ca2+-activated K+ channels (SKCa, IKCa and BKCa, respectively) have been implicated, with inward rectifier K+ channels (KIR) and Na+/K+-ATPase also suggested by some studies. 2. Endothelium-dependent membrane currents recorded using single-electrode voltage-clamp from electrically short lengths of arterioles in which the smooth muscle and endothelial cells remained in their normal functional relationship have provided useful insights into the mechanisms mediating EDHF. Charybdotoxin (ChTx) or apamin reduced, whereas apamin plus ChTx abolished, the EDHF current. The ChTx- and apamin-sensitive currents both reversed near the expected K+ equilibrium potential, were weakly outwardly rectifying and displayed little, if any, time- or voltage-dependent gating, thus having the biophysical and pharmacological characteristics of IKCa and SKCa channels, respectively. 3. The IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells. There is little evidence for a significant number of these channels in healthy, contractile vascular smooth muscle cells. 4. In a number of blood vessels in which EDHF occurs, the endothelial and smooth muscle cells are coupled electrically via myoendothelial gap junctions. In contrast, in the adult rat femoral artery, in which the smooth muscle and endothelial layers are not coupled electrically, EDHF does not occur, even though acetylcholine evokes hyperpolarization in the endothelial cells. 5. In vivo studies indicate that EDHF contributes little to basal conductance of the vasculature, but it contributes appreciably to evoked increases in conductance. 6. Endothelium-derived hyperpolarizing factor responses are diminished in some diseases, including hypertension, pre-eclampsia and some models of diabetes. 7. The most economical explanation for EDHF in vitro and in vivo in small vessels is that it arises from the activation of IKCa and SKCa channels in endothelial cells. The resulting endothelial hyperpolarization spreads via myoendothelial gap junctions to result in the EDHF-attributed hyperpolarization and relaxation of the smooth muscle.     

Effect of methomyl on hepatic mixed function oxidases in rats

Objective:

To study the effect of the methomyl on mixed function oxidase system in rats.

Materials and Methods:

The effect of the methomyl on mixed function oxidase was studied using different dosages, durations and sex. Microsomes were isolated using the calcium precipitation method. The levels of cytochrome P₄₅₀ , and cytochrome b₅ were determined using extinction coefficient of 91 and 85 mM^-1 respectively. The activities of drug metabolizing enzymes, hemoglobin content, liver function enzymes, and serum cholinesterase activity were assayed by using standard methods.

Results:

Intraperitoneal administration of methomyl (4 mg/kg body weight) showed significant decrease in the level of cytochrome P₄₅₀ , and the activities of aminopyrine N-demethylase and aniline hydroxylase on the third day of the treatment. Methomyl (4 mg/kg) treatment of old male rat and adult female rat also showed a decrease in the level of cytochrome P₄₅₀ , and aminopyrine N-demethylase activity. The serum samples from methomyl treated rats (male and female), when analyzed for alanine aminotransferase (SGPT) and aspartate aminotransferase (SGOT) as markers of the liver toxicity, showed significant increase in the activity. The activities of SGPT and SGOT were significantly higher in the treated rats (2 and 4 mg/kg) than in the control group. A significant decrease in the level of hemoglobin and serum cholinesterase activity was observed, when there was an increase in the dose level. A significant increase was observed in alkaline phosphatase activity at all dose levels.

Conclusion:

Methomyl influences mixed function oxidase and creates abnormality of liver functions in the rats. This effect depends on the dose and duration of methomyl.     

Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells.
2. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (G_max) of 27.4±1.4 nS and a mean −log EC₅₀ of 5.12±0.03 (mean±s.e.mean) (n=114).
3. Muscarinic antagonists with higher affinity for the M₂ receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA₂ values of 8.1, 8.0 and 9.1, respectively.
4. All M₃ selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC₅₀ for carbachol. At higher concentrations M₃ antagonists shifted the agonist curve to the right, increasing the EC₅₀, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in G_max (M₃ effect) and significant increase in the EC₅₀ (M₂ effect) in the same concentration range.
5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected.
6. The results support the hypothesis that carbachol activates M₂ muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M₃ receptors. As an increasing fraction of M₃ receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M₂ receptors are disabled.

     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

Actions of the novel thromboxane A2 antagonists,ONO-1270 and ONO-3708, on smooth muscle cells of the guinea-pig basilar artery

Summary The effects of the novel thromboxane A₂ (TXA₂) antagonists. ONO-1270 and ONO-3708, on the electrical and mechanical responses evoked by various agents, and in particular 9, 11-epithio-11, 12-methano-thromboxane A₂ (STA₂), were investigated in the guinea-pig artery. STA₂ (up to 0.3 M), and ONO-1270 and ONO-3708 (up to 1.0 M) dit not modify the membrane potential in smooth muscle cells. Perivascular nerve stimulation induced an excitatory junction potential (e.j.p.), and with frequencies over 0.25 Hz, depression of e.j.ps occurred. STA₂ (0.1 M) and both ONO-1270 and ONO-3708 had no effect on these electrical events. STA₂ (over 0.1 M) produced phasic and tonic contractile responses, in a concentration dependent manner. Both ONO-1270 and ONO-3708 competitively inhibited the phasic contraction induced by STA₂ as estimated from parallel shifts in the dose-response curve, and from the Lineweaver-Burk and Schild plots (the PA₂ values were 8.22 for ONO-1270 and 8.70 for ONO-3708), but both agents inhibited non-competitively the PGF₂ -induced contraction. ONO-1270 and ONO-3708 (up to 0.1 M) had no effect on contractions induced by K⁺ and caffeine, but did slightly inhibited contractions induced by 5-hydroxytryptamine (5-HT). Following application of indomethacin, neither agent modified the 5-HT-induced contraction. In Ca²⁺-free solution, 10 nM STA₂ produced a phasic but not a tonic contractile response. ONO-1270 and ONO-3708 (over 1 nM) inhibited this phasic contractile response. We conclude that ONO-1270 and ONO-3708 possess the properties of potent and selective antagonists for the TXA₂ (STA₂)-receptor in smooth muscle cells of the guinea-pig basilar artery.     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

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.

     

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.     

The interaction of diadenosine polyphosphates with P2X-receptors in the guinea-pig isolated vas deferens

1. The site(s) at which diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP₄A) and diadenosine 5′, 5′′′-P¹,P⁵-pentaphosphate (AP₅A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P₂-receptor antagonists and the ecto-ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal-5′-phosphate (P-5-P) (3–1000 μM) inhibited contractions evoked by equi-effective concentrations of AP₅A (3 μM), AP₄A (30 μM) and α,β-methyleneATP (α,β-meATP) (1 μM), in a concentration-dependent manner and abolished them at the highest concentrations used.
3. PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP₅A, AP₄A and α,β-meATP with similar IC₅₀ values. No significant difference was found between IC₅₀ values for suramin against α,β-meATP and AP₅A or α,β-meATP and AP₄A, but suramin was more than 2.5 times more potent against AP₄A than AP₅A. P-5-P showed the same pattern of antagonism.
4. Desensitization of the P_2X1-receptor by α,β-meATP abolished contractions evoked by AP₅A (3 μM) and AP₄A (30 μM), but had no effect on those elicited by noradrenaline (100 μM).
5. ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP₄A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP₅A (3 μM).
6. It is concluded that AP₄A and AP₅A act at the P_2X1-receptor, or a site similar to the P_2X1-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP₄A, but not AP₅A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.

     

Action of a novel potassium channel opener,SR 47063, on human bronchi and on guinea-pig trachea in vitro: comparison with cromakalim

Potassium channels are present on airway smooth muscle cells and their activation results in hyperpolarization and relaxation. Because these effects may have therapeutic relevance to asthma, the aim of our study was to examine the activity of SR 47063, a potassium channel opener (KCO), against a variety of spasmogens or against electrical field stimulation in guinea-pig isolated trachea and in human isolated bronchi in vitro; the effects of SR 47063 were compared with those of cromakalim, isoprenaline, and theophylline. Like cromakalim, SR 47063 reduced the contractility of guinea-pig isolated trachea and the human isolated bronchus in basal tone with pD₂ of 7.79 ± 0.01 and 7.83 ± 0.09, respectively, or during precontractions induced by acetylcholine 10^?4 M, histamine 10^?5 M, or low concentrations of KCl (<30 mM), but not by high KCl concentrations (≥30 mM); these effects were antagonized by glibenclamide 10^?5 M. This spectrum of action is typical of the compounds known as potassium channel openers. Electrical field stimulation (EFS: 16 Hz, 1 ms, 320 mA for 10 sec in the presence of indomethacin 10^?6 M and propranolol 10^?6 M) of guinea-pig isolated main bronchi induced 2 successive contractile responses. Both contractions were reduced significantly by SR 47063 and cromakalim. Although we have not studied the effects of KCOs on exogenous neurokinin A- or substance P-induced contractions, it might be suggested as a hypothesis that this inhibition seems to take place presynaptically and to affect the release of neuromediators produced by electrical field stimulation. In conclusion, SR 47063 exerts in vitro on the bronchial smooth muscle an inhibitory effect which seems to be due to the opening of glibenclamide-sensitive potassium channels. SR 47063 is 3- to 10-fold more potent than cromakalim. © 1993 Wiley-Liss, Inc.     

Inhibitors of spasmogen-induced Ca2+ channel suppression in smooth muscle cells from small intestine

1. Whole-cell patch-clamp recordings were made from smooth muscle cells isolated from the longitudinal muscle layer of guinea-pig ileum. Carbachol (acting at muscarinic receptors) or histamine (acting at H₁ histamine receptors) suppressed Ca²⁺ channel current. The effect of either agonist had an initial transient component followed by a sustained component.
2. Wortmannin inhibited transient and sustained components of carbachol-induced Ca²⁺ channel current suppression: half-effective inhibitory concentrations (IC₅₀) were 1.1 μM and 0.6 μM for the two components respectively. Wortmannin also inhibited the transient phase of carbachol-induced cationic current (IC₅₀ 1.6 μM) and Ca²⁺-dependent K⁺-current (IC₅₀ 1.7 μM). Wortmannin did not appear to produce any direct block of cationic channels or Ca²⁺ channels.
3. Intracellular application of the phospholipase inhibitor D609 (tricyclodecan-9-ylxanthogenate) inhibited transient and sustained components of histamine action on the Ca²⁺ channel current: the IC₅₀ was about 130 μM for both components. Carbachol action on Ca²⁺ channels was also inhibited by D609. D609 had no significant direct blocking effect on Ca²⁺ channels, cationic channels activated by carbachol, or Ca²⁺-activated K⁺-current in response to flash-photolysis of caged-inositol 1,4,5-trisphosphate.
4. Micromolar concentrations of wortmannin and D609 are inhibitors of both components of spasmogen-induced Ca²⁺ channel suppression. The data suggest that both components are mediated by a common, or similar, signal transduction element which is a phospholipase C (PLC) or phospholipase D (PLD) isoform.