Effect of potassium channel modulators on toxicity of Cleistanthus collinus

The study was conducted to determine the effects of boiled extract of Cleistanthus collinus on rats by observing ECG changes and electrolyte levels in serum and urine. Influence of minoxidil and glibenclamide on Cleistanthus collinus induced toxicity was determined. ED50 for arrhythmia, changes in contractility and heart rate were recorded using the isolated frog heart. Cleistanthus at low doses caused transient tachycardia and increase in contractility and at high dose caused arrhythmia and cardiac arrest in rat. LD50 was found to be 1690 mg/kg. Minoxidil potentiated cardiac toxicity, whereas glibenclamide did not produce any significant change. High concentration of potassium in Cleistanthus extract hindered comparison of its levels. There was excretion of sodium even in the presence of hyponatraemia. Cleistanthus at low dose caused transient tachycardia and increase in contractility and at high dose caused arrhythmia and cardiac arrest in isolated frog heart. ED50 for arrhythmia was found to be 1406 mg/kg. Acute toxicity was mainly due to depressive cardiac activity of Cleistanthus. It also caused renal failure. Potassium channel modulators did not have important role in acute cardiac toxicity treatment. Probably in chronic toxicity, electrolyte level changes are involved and potassium channel modulators might have a role.     

Effect of ATP-sensitive potassium channel modulators and intermittent hypoxia on mitochondrial respiration during stress

The influence of activator of ATP-sensitive potassium channels (KATP) pinacidil and blocker glibenclamide after intermittent hypoxia in rats under stress condition on ADP-stimulated mitochondrial respiration by Chance and lipid peroxidation processes in liver have been investigated. We used next substrates of oxidation--0.35 mM succinate, 1 mM alpha-ketoglutarate, 3 mM glutamate, 3 mM pyruvate, 2.5 mM malate and inhibitor of the mitochondrial fermentative complex I (10 microM rotenone), succinate dehydrogenase inhibitor (2 mM malonate) and inhibitor of transamination (1 mM aminooxiacetate). We suggest that adaptation by intermittent hypoxia and application of a KATP opener pinacidil possess significant protective effect on mitochondrial energy support under stress condition. Combination of intermittent hypoxia with pinacidil causes more efficient consumption of oxygen and decrease of lipid peroxidation processes comparative to intermittent hypoxia or pinacidil injection used separately. We conclude about the existence of the functional link between nitric oxide which is being increased under intermittent hypoxia and KATP opener. Both intermittent hypoxia and pinacidil effectively decrease the negative results of mitochondrial dysfunction under stress condition.     

Contractile effects of vanadate on aorta rings from virgin and pregnant rats

The present study was undertaken to characterize the contractile effects of vanadate on thoracic aorta rings from virgin and term-pregnant rats. Vanadate caused concentration-dependent contraction in rat aortic rings with an EC₅₀ (concentration producing 50% maximum response) of 0.10 mM. Contractions in response to vanadate were equivalent to the ones measured with 1 M phenylephrine. The effects of vanadate were not affected by indomethacin (up to 10 M), an inhibitor of prostanoid cyclooxygenase, but were blocked in a concentration-dependent manner by staurosporine (0.1–1.0 M), an inhibitor of protein kinase C. Vanadate exhibited a significant decrease of contractile responses in aorta of pregnant as compared to virgin rats. When aortic rings were bathed in presence of different concentrations of vanadate, the concentration-response curve to phenylephrine was shifted to the left, but maximum response was not affected. The potentiation of the contractions to phenylephrine by vanadate was significantly more prominent in aorta of virgin than of pregnant rats. These results suggest that the contractile effect of vanadate on rat aorta is independent of endogenous prostanoids and may be mediated by protein kinase C-dependent pathway. These results also show that the contractile response to vanadate on the rat aorta is impaired during pregnancy.     

Modulation of calcium mobilization in aortic rings of pregnant rats: Contribution of extracellular calcium and of voltage-operated calcium channels

Pregnancy is associated with decreased vascular responsiveness to vasopressor stimuli. We have tested the involvement of Ca2+ mobilization in myotropic responses of aortic rings obtained from pregnant and virgin rats. Contractions of the rings to phenylephrine, in the absence of calcium in the bathing medium, were lower in tissues from virgin than from pregnant rats. Concentration-response curves to CaCl2 that were measured after stimulation by phenylephrine in the absence of Ca2+ were shifted to higher levels of contraction. This was not observed when KCl was used to prestimulate the aorta. D-600, a phenylalkylamine calcium channel blocker, similarly inhibited these responses to CaCl2 in tissues from both pregnant and virgin animals. D-600 exerted a concentration-dependent inhibition of responses to phenylephrine and KCl. However, the calcium antagonist was less effective in aortic rings of pregnant than of virgin rats. Basal 45Ca2+ uptake was lower in aortic rings from pregnant than from virgin rats, and Bay K 8644 was unable to reverse this difference. The time course of basal and stimulated (KCl) 45Ca2+ influx was lower in aorta of pregnant rats at all times studied. Moreover, when the intracellular calcium pools were emptied with phenylephrine, the refilling of these pools was delayed in aortic rings of pregnant rats. These results indicate an altered extracellular calcium mobilization of aortic rings from pregnant rats. These changes may be due to a functional alteration of the voltage-operated calcium channels during pregnancy.     

Effects of calcium channel blockers on potassium homeostasis.

The known effects of calcium channel blockers on various aspects of potassium homeostasis are reviewed. Regulation of potassium homeostasis requires both renal and external handling mechanisms. Signaling by calcium appears to mediate both of these. Calcium channels have been identified in adrenal glomerulosa cells, and cellular calcium entry has been demonstrated in vitro to be necessary for the synthesis and secretion of aldosterone. Calcium channel antagonists such as verapamil and nifedipine, at pharmacologic doses, can block aldosterone production. In vivo, however, only chronic administration of verapamil appears to attenuate aldosterone responsiveness to angiotensin II. Chronic administration of nifedipine does not have a dramatic effect on aldosterone production following potassium loading. Other studies have demonstrated improved extrarenal potassium disposal following treatment with calcium channel blocking agents. Clinically, there are no reports of either hyperkalemia or hypokalemia with the routine therapeutic use of these agents given alone. This review was prompted by the development of hyperkalemia in a patient with chronic renal failure following the initiation of therapy with the calcium channel blocker diltiazem: however, numerous other etiologies may also have contributed to the development of hyperkalemia in this case. Review of the data indicates that current evidence implicating this class of drugs in the pathogenesis of disordered potassium regulation remains equivocal.     

Effects of anandamide on gestation in pregnant rats

The present study was to test whether the recently described endogenous ligand for the cannabinoid receptor; arachidonyl-ethanolamide (anandamide, ANA), may produce similar effects on pregnancy as the main psychoactive component of marihuana: Δ⁹-tetrahydrocannabinol (THC) in rats. ANA, THC (0.02 mg/kg i.p./day, respectively) or vehicle were injected daily over the third week of pregnancy. The pregnant rats were either killed on day 21 of pregnancy or followed up to delivery. Results show a significant increase in the duration of pregnancy after both THC and ANA treatment. Both drugs caused an increase in the frequency of stillbirths. The mothers' hormone contents in tissues and sera were measured. Decreased LH content was observed in the serum of treated animals. No changes in FSH content were observed either in the pituitary or in the sera. Pituitary prolactin (PRL) levels was lower in ANA treated animals as compared both to controls or THC treated subjects. The serum PRL content decreased in all experimental groups. Decrease in serum progesterone was more prominent in treated rats. Serum levels of prostaglandins (PGF 1 and PGF 2) were significantly decreased after THC and ANA treatment. We conclude that ANA has the same tendency to change reproductory parameters in pregnant rats as THC, although in some cases the effects of ANA were slightly different from that of THC. Both endogenous and exogenous cannabinoids inhibit PG synthesis in pregnant rats and this maybe responsible for the delay constitute the mechanism in the onset of labour.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells

Potassium channels have been identified as targets for a large number of therapeutic indications. The ability to use a high-throughput functional assay for the detection and characterization of small-molecule modulators of potassium channels is very desirable. However, present techniques capable of screening very large chemical libraries are limited in terms of data quality, temporal resolution, ease of use, and requirements for specialized instrumentation. To address these issues, the authors have developed a fluorescence-based thallium flux assay. This assay is capable of detecting modulators of both voltage and ligand-gated potassium channels expressed in mammalian cells. The thallium flux assay can use instruments standard to most high-throughput screening laboratories, and using such equipment has been successfully employed to screen large chemical libraries consisting of hundreds of thousands of compounds.     

ATP敏感性钾通道开放剂埃他卡林对大鼠低氧性肺动脉高压的影响

目的 :探讨新型ATP敏感性钾通道开放剂 (KATPCO)埃他卡林 (iptkalim ,Ipt)对低氧性肺动脉高压 (HPH)大鼠肺血管重构的影响。方法 :将大鼠置于常压低氧舱内 (O2 1 0 %± 0 .5 % ) ,8h/d ,每周 6d ,4周后测定平均肺动脉压(mPAP)、RV/ (LV +S) ;用图象分析仪测量与呼吸性细支气管伴行的肺小动脉外径 (ED)、动脉中层壁厚 (MT)、动脉管壁中层面积 (MA)、动脉管腔面积 (VA)和血管总面积 (TAA)。结果 :慢性低氧组大鼠的mPAP和RV/ (LV +S)显著高于正常对照组 (P <0 .0 1 ) ;图象分析显示低氧组大鼠肺小动脉中层壁厚与动脉外径百分比 (MT % )、动脉壁中层面积与血管总面积百分比 (MA % )均显著高于对照组 (P <0 .0 1 ) ;慢性低氧组大鼠肺小动脉管腔面积 (VA)与血管总面积 (TAA)百分比显著低于正常组 (P <0 .0 1 )。Ipt 0 .75mg·kg- 1 ·d- 1 和 1 .50mg·kg- 1 ·d- 1 均可显著抑制低氧性肺血管壁重构 ,降低肺动脉压 ,减少右心室肥厚 ,1 .50mg·kg- 1 ·d- 1 则可逆转持续低氧所致的所有病理性变化。结论 :新型KATPCO埃他卡林是一个富有潜力的治疗HPH药物     

Inhibitory effects of potassium channel blockers on tetramethylpyrazine-induced relaxation of rat aortic strip in vitro

Tetramethylpyrazine (TMP) is one of the active principles contained in Ligusticum chuanxiong Hort. (Umbelliferae), a herb that has been widely used to treat vascular disorders in China. In the present study, role of potassium channel in the vasodilatation of TMP was investigated using the effect of potassium channel blocker on TMP induced relaxation in isolated aortic rings from Wistar rats. TMP produced a concentration-dependent relaxation in the aortic rings precontracted with vasopressin or phenylephrine. Similar effect of TMP on vasoconstrictions by phenylephrine and vasopressin, induced through two different receptors, indicating the direct vasodilatation of TMP. Specific inhibitors for potassium channel were used to characterize the role of potassium channel in this action of TMP. Only the inhibitors specific to small conductance calcium-activated potassium (SK(Ca)) channel or ATP-sensitive potassium (K(ATP)) channel inhibited the action of TMP. Also, the TMP-induced relaxation was reversed by the inhibitor of soluble guanylyl cyclase in a way similar to that of K(ATP) channel blockade. The obtained results indicated that vasodilatation induced by TMP is related to the opening of SK(Ca) and K(ATP) channels.     

Effects of angiotensin II on isolated toad (Bufo arenarum) aortic rings

1. The vascular response to Asn1-Val5 angiotensin II (A II) in aortic rings from Bufo arenarum toad was studied. 2. Tachyphylaxis in response to A II could be abolished after incubation with norepinephrine (NE). 3. Phentolamine treatment partially inhibited the pressor effects to A II. 4. Sar1-Ile8 A II and Sar1-Ala8 A II significantly attenuated the vascular effects of A II and did not affect the NE response. 5. We conclude that the pressor response to A II has a direct contractile effect and a catecholamine dependent component in aortic rings of Bufo arenarum toad.     

Effects of modulators of N-methyl-D-aspartate receptor-mediated neurotransmission on diazepam discrimination in rats

N-methyl-D-aspartate (NMDA) antagonists share a number of pharmacological effects with GABA(A) agonists, including anxiolytic and anticonvulsant effects. This study evaluated the effects of site-selective NMDA antagonists in rats trained to discriminate the benzodiazepine diazepam from vehicle. As expected, diazepam produced robust discriminative stimulus effects and dose-dependently substituted for the training dose. Mixed results were obtained with competitive NMDA antagonists: whereas NPC 17742 partially substituted for diazepam, SDZ EAA 494 did not elicit responding on the diazepam-associated lever. Other site-selective NMDA antagonists, including the open channel blocker phencyclidine, the glycine-site antagonists ACEA 1021 and MDL 102,288, the polyamine-site antagonist arcaine, and the glutamate release inhibitor riluzole, failed to substitute for diazepam. Agonists at nonbenzodiazepine sites of the GABA(A) receptor complex were also tested for comparison purposes. The barbiturate pentobarbital and the neurosteroid Co 2-1068 partially substituted for diazepam. In contrast, the anticonvulsant carbamazepine failed to substitute even at a dose that substantially reduced response rates. These results suggest that substitution of NMDA antagonists for GABA(A) agonists is dependent upon the site at which the NMDA antagonist binds. Further, they suggest that similarities between the stimulus properties of GABA(A) agonists and NMDA antagonists are at least as strong as similarities among agonists acting at different sites on GABA(A) receptors.     

Dependence of media cross-sectional area on circumference of aortic rings from normal and hypertensive rats

The purpose of this study is to determine if cross-sectional area and media thickness depend on circumference in excised vessels. Measurements were obtained from rat aortic rings chemically fixed after excision. Rings were fixed without distention or after mounting on circular rods of different diameters. Media cross-sectional area increased with circumference but thickness decreased. Thickness and area were significantly greater in the SHR than the WKY rat at all circumferences. Results of a theoretical analysis predict that cross-sectional area and thickness will depend on circumference in excised rings. Best-fit curves of the experimental data to the theoretical relationships show that neither thickness nor cross-sectional area of the aorta is consistently better correlated with circumference in SHRs and WKY rats. The following conclusions are made: (i) Media cross-sectional area and thickness depend on circumference in excised-aortic rings, and (ii) for a specific method of preparation, thickness and cross-sectional area are equally accurate when comparing aortic rings from normal and hypertensive rats. If an experimental determination of the relationship between circumference and cross-sectional area (or thickness) cannot be done, we suggest that a reasonable alternative is to compare these measurements at the same circumference.     

Myocardial infarction increases reactivity to phenylephrine in isolated aortic rings of ovariectomized rats

Clinical studies demonstrated that the incidence of cardiovascular disease is low in premenopausal women, rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy. The interaction between gender and myocardial infarction indicates that the survival advantage of women is modified by the occurrence of myocardial infarction. Therefore, the effect of myocardial infarction on mortality is greater in women than men. The aim of our study was to investigate the influence of the ovariectomy on the reactivity to phenylephrine in aortic rings of female rats post-myocardial infarction. Animals were divided in four groups: Control (Cont), Ovariectomized (Ovx), Infarcted (Inf) and Ovariectomized and Infarcted (Ovx-Inf). Aortic rings were studied 60 days after ovariectomy and infarction surgery. The infarct area was similar among groups. The maximal response to phenylephrine was increased in the Ovx-Inf group compared to all the other groups (Cont = 2.411+/-0.131 (N = 11); Ovx = 2.863+/-0.121(N = 15); Inf = 2.794+/-0.102 (N = 13); Ovx-Inf = 3.40+/-0.201* (N = 12) g; *P < 0.05). In the absence of endothelium and L-NAME perfusion, the maximal response to phenylephrine was similarly increased in all groups. Relaxation to acetylcholine was also similar. The indirect evaluation of NO bioavailability analyzed by the area under the curve demonstrated a reduction on NO on the Ovx-Inf group that could contributes to increased response to phenylephrine. In conclusion our results showed that ovariectomy associated to a myocardial infarction leads to an increment of aorta reactivity to phenylephrine associated to a reduction of basal NO bioavailability in spite of a normal endothelium-dependent relaxation induced by acetylcholine.     

Synthesis and activity on rat aorta rings and rat pancreatic beta-cells of ring-opened analogues of benzopyran-type potassium channel activators

Ring-opened analogues of dihydrobenzopyran potassium channel openers (PCOs) were prepared and evaluated as putative PCOs on rat aorta rings (myorelaxant effect) and rat pancreatic beta-cells (inhibition of insulin secretion). These derivatives are characterized by the presence of a sulfonylurea, a urea or an amide function. Some compounds bearing an arylurea moiety provoked vasorelaxant effects and a marked inhibition of insulin release. Derivatives bearing a sulfonylurea or an amide function were, however, poorly active on both tissues. Structure-activity relationships and apparent tissue selectivity are discussed.     

Effect of calcium channel modulators on isolated endothelial cells

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.     

Partial structures of ketoconazole as modulators of the large conductance calcium-activated potassium channel (BK(Ca))

A series of partial structures of ketoconazole has been synthesized and tested for activity on the large conductance calcium-activated potassium channel (BK) in bovine smooth muscle cells. This has provided openers and blockers of the channel. The results suggest that the phenyl and phenoxy moieties are important for interaction with BK, whereas the imidazole group is unimportant. The properties of the phenoxy moiety seem to determine whether the compounds act to open or block the channel.