Pharmacological evidence for calcium channel inhibition by danshen (Salvia miltiorrhiza) on rat isolated femoral artery

This study investigated the relaxant actions of danshen (Salvia miltiorrhiza) and its lipid-soluble- and water-soluble-fractions on endothelium-denuded rat isolated femoral artery rings. Danshen, its water-soluble fraction and its lipid-soluble fraction produced relaxation of the phenylephrine-precontracted artery rings with IC50 values of 149 +/- 20 microg/mL, 160 +/- 25 microg/mL, and 23 +/- 6 microg/mL, respectively. Pretreatment of the artery rings with a non-selective potassium channel inhibitor tetraethylammonium (TEA, 10 mM) produced a significant two-fold rightward shift of the concentration-response curve to danshen and a four-fold shift to its water-soluble fraction, but had no effect on the lipid-soluble fraction. A 3.3-fold shift was produced on the concentration-response curve of danshen when the artery rings were pretreated with a mixture of 10 mM TEA, 1 mM 4-aminopyridine (K(V) blocker), 1 microM glibenclamide (K(ATP) blocker), 100 nM iberiotoxin (BK(Ca) blocker), and 100 microM barium chloride (K(IR) blocker). Involvement of Ca2+ channels was investigated in endothelium-denuded artery rings incubated with Ca2+-free buffer and primed with 1 microM phenylephrine or 60 mM KCl for 5 minutes prior to adding CaCl2 to elicit contraction. In artery rings primed with phenylephrine, pretreatment with 1 mg/mL danshen, 1 mg/mL water-soluble fraction of danshen, 0.1 mg/mL lipid-soluble fraction of danshen, and 100 nM nifedipine abrogated the CaCl2-induced contraction. On the other hand, in artery rings primed with KCl, these agents produced 40%, 25%, 53%, and 92% inhibition on the maximum contraction induced by CaCl2, respectively. Increasing the concentrations of danshen and its water-soluble fraction to 3 mg/mL, and the lipid-soluble fraction to 0.3 mg/mL further reduced the maximum contraction to 92%, 93%, and 83%, respectively. Taken together, these findings suggested the vasorelaxant actions of danshen and its fractions were produced primarily by inhibition of Ca2+ influx in the vascular smooth muscle cells and a small component was mediated by the opening of K+ channels.     

Relaxant effects of danshen aqueous extract and its constituent danshensu on rat coronary artery are mediated by inhibition of calcium channels

In this study, we have investigated the actions of danshensu, an active, water-extractable component of the medicinal herb danshen (Salvia miltiorrhiza), on rat isolated coronary artery rings precontracted with 1 microM 5-hydroxytryptamine (5-HT) and its action compared to the water-extractable fraction of the herb. Extraction of the water-soluble fraction from danshen (S. miltiorrhiza) provided yield of 17.5% (35 g/200 g). The amount of danshensu determined in the crude danshen herb and in its aqueous fraction was 0.45 mg/g (0.045%) and 3.28 mg/g (0.33%). The danshen aqueous extract was 13 times less potent than danshensu in relaxing 5-HT-precontracted coronary artery rings; IC50 values were 930.3+/-133.5 microg/ml and 71.5+/-11.0 microg/ml. Removal of the endothelium did not significantly affect their vasodilator potencies; IC50 values were 842.1+/-123.8 microg/ml and 84.8+/-8.8 microg/ml. On the other hand, a potassium channel inhibitor tetraethylammonium (TEA, 10 mM) shifted their concentration-response curves by 1.7 and 2.2 folds. The possible involvement of Ca2+ channels was investigated in artery rings incubated with Ca2+-free buffer and primed with 1 microM 5-HT or 60 mM KCl for 5 min prior to addition of CaCl2 to elicit contraction. In 5-HT-primed preparations, the CaCl2-induced vasoconstriction was abolished by 2 mg/ml danshen aqueous extract and 200 microg/ml danshensu, whereas, in KCl-primed preparations, 10 mg/ml danshen aqueous extract and 600 microg/ml danshensu were required to abrogate the vasoconstriction. These findings suggest the vasorelaxant actions of danshen aqueous extract and danshensu were produced by inhibition of Ca2+ influx in the vascular smooth muscle cells. The opening of K+ channels had a minor contribution to the response, but endothelium-dependent mechanisms were not involved.     

丹参在炮制大生产过程中丹酚酸B的损失情况研究

目的考察丹参药材炮制大生产过程中指标性成分丹酚酸B的含量变化及损耗情况。方法进行10批次丹参饮片炮制生产,应用HPLC法,在生产过程各主要工艺点进行跟踪取样,测定丹酚酸B的含量并计算损耗;同时在其中一个批次药材炮制大生产过程中,检测每个工艺环节对丹酚酸B的含量的影响。结果原药材、水洗药材、润透药材和切片4个取样点丹酚酸B的平均含量分别为6.83%,4.72%,4.46%和3.55%,相对原药材的损失情况分别为30.84%,34.59%和47.99%。水洗工艺直接导致丹酚酸B损失22.11%,最终损失高达47.48%。结论严格控制(优化)水洗工艺,可有效保留丹酚酸B的含量,提高丹参的炮制效率。     

Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat

We assessed the bioavailability of magnesium lithospermate B (MLB), a main polyphenolic component of Salvia miltiorrhiza and a potent antioxidant having various pharmacological activities, to evaluate its action in vivo. The plasma concentrations of lithospermic acid B (LSB) showed a biexponential decrease after intravenous administration of MLB to rats at doses of 4 and 20 mg/kg. The values of area under the concentration-time curve (AUC; 87.8 +/- 10.9 and 1130 +/- 329 microg.min/mL), total body clearance (CL (tot); 55.52 +/- 7.07 and 23.51 +/- 5.98 mL/min/kg), and distribution volume at steady state (V (ss); 7.60 +/- 1.03 and 3.61 +/- 1.16 L/kg) suggested non-linear pharmacokinetics between the two doses. After oral administration of MLB at a high dose of 100 mg/kg, the mean AUC was barely 1.26 +/- 0.36 microg.min/mL. Absolute bioavailability of MLB was calculated to be 0.0002 from the AUC values after both intravenous dosing at 20 mg/kg and oral dosing at 100 mg/kg. The extremely low bioavailability was caused mainly by poor absorption from the rat gastrointestinal tract; about 65 % of the dose was retained in the tract even 4 h after oral administration, and most of the dose was retained even 20 min after infusion in an in situ jejunal loop experiment. Urinary and biliary excretion of LSB were only 0.70 % +/- 0.26 % and 5.10 % +/- 2.36 %, respectively, over a 30 h time period after intravenous injection despite the large CL (tot) and V (ss) values, and were much less (0.010 % +/- 0.001 % and 0.12 % +/- 0.04 %) after oral dosing. These findings suggest that extensive metabolism, including a first-pass effect, and wide distribution of LSB besides the poor absorption contributed significantly to the extremely low systemic bioavailability.     

正交试验优选丹参中丹酚酸B的提取工艺

目的:优选丹参中丹酚酸B的提取工艺。方法:以丹酚酸B含量及得率为指标,乙醇浓度、乙醇倍数、回流提取次数、提取时间为因素进行正交试验,优选丹参中丹酚酸B的最佳提取工艺。结果:最佳提取工艺为5倍的80%乙醇提取2次,每次1h,可得29.13%的丹酚酸B,得率为6.14%。结论:该方法操作简便、结果稳定,可为工业生产提供理论依据。     

HPLC-MS法测定白花丹参中丹酚酸B的含量

目的:建立以高效液相色谱-质谱法测定白花丹参中丹酚酸B含量的方法。方法:色谱柱为Symmetry field RP C_(18) (150mm×2.1mm,3.5μm),流动相为甲醇水,流速为0.2mL·min~(-1),检测波长为285nm;质谱为Waters micromass ZQ4000质谱仪,离子模式为选择离子(SIR)。结果:丹酚酸B在0.05～60μg·mL~(-1)浓度范围内与峰面积积分值呈良好线性关系(r=0.999 8);平均加样回收率为99.18%,RSD=0.44%(n=3)。结论:本方法可为白花丹参的质量评价和道地药材质量标准的建立提供理论依据。     

15,16-Dihydrotanshinone I, a major component from Salvia miltiorrhiza Bunge (Dansham), inhibits rabbit platelet aggregation by suppressing intracellular calcium mobilization

Radix Salviae miltiorrhiza (RSM, ‘Dansham’ in Korea, ‘Danshen’ in Chinese), the root of Salviae miltiorrhiza Bunge (Labiate) has been used as Chinese fork medicine for the treatment of cardiovascular diseases such as angina pectoris, coronary heart disease, myocardial infarction, and hypertension. In the present study, we evaluated the inhibitory effects of 15, 16-Dihydrotanshinone I, one of the major ingredients of Salvia miltiorrhiza Bunge, on platelet aggregation, with elucidation of its mechanisms of action. 15,16-Dihydrotanshinone I concentration-dependently inhibited collagen-induced aggregation of rabbit washed platelets with IC₅₀ of 8.7±5.6 μM, the potency being about seven-fold greater than EGCG, an active Green tea catechin component (IC₅₀: 56.6±48.7 μM). 15,16-Dihydrotanshinone I significantly inhibited the intracellular calcium ([Ca²⁺]_i) mobilization in a concentration-dependent manner. 15,16-dihdydrotanshinone I also significantly suppressed collagen (50 μg/mL)-induced liberation of [³H]Arachidonic acid from [³H]Arachidonic acid-incorporated rabbit platelet. In addition, 15,16-Dihydrotanshinone I at 50 μM slightly but significantly inhibited collagen-induced production of thromboxane B₂. These results indicate that 15,16-Dihydrotanshinone I exert potent anti-platelet activity via suppression of [Ca²⁺]_i mobilization and arachidonic acid liberation.     

HPLC法测定复方丹参微丸中丹酚酸B的含量

目的：用高效液相色谱法测定复方丹参微丸中丹酚酸B的含量。方法以乙腈-甲醇-甲酸-水（10∶30∶1∶59）为流动相,色谱柱 Diamon-silTM C18（5μm,4.6＆#215;250mm）,波长286nm、柱温40℃、流速1.0mL＆#183; min -1为色谱条件,对复方丹参微丸中丹酚酸 B含量进行分析。结果丹酚酸B在59.5～1487.5 ng范围内成良好的线性关系（ r＝0.99995）。结论本法简便、准确、重现性好。     

盐酸水提法制备丹参水溶性有效部位群及其对成骨细胞活性测定

目的探讨丹参水溶性有效部位群(salvia miltiorrhiza utility aqueous extract,DUAE,丹参骨宝2号)的制备工艺,质量控制及其对成骨细胞活性测定。方法采用盐酸水提法处理丹参药材,制得丹参骨宝2号,用高效液相法(HPLC)进行含量测定;通过PNPP检测法比较DUAE对SD大鼠成骨细胞(osteoblast,OB)的碱性磷酸酶(ALP)表达水平的影响,评价丹参骨宝2号药物机制。结果用盐酸水提法制备的丹参骨宝2号与传统水提方法比较,丹参素含量提高3·28倍,为(11·50±0·52)mg·g-1生药,丹酚酸B含量减低1·46倍,为(16·40±1·22)mg·g-1生药,且两者含量均在1%~1·5%,原儿茶醛含量增加1·46倍,为(0·233±0·02)mg·g-1生药。传统水提法制备的DUAE药量为1mg生药·L-1时仅提高成骨细胞ALP活性5·5%±0·3%。丹参骨宝2号药量为0·25mg生药·L-1时提高成骨细胞ALP活性达52%±10%。结论用盐酸水提法制备的丹参骨宝2号中含有较高的丹参素和一定比例的丹酚酸B、原儿茶醛,且成分含量稳定,质量可控,对成骨细胞活性测定显示药效好,为研究丹参抗骨质疏松新制剂提供有益资料。     

Mechanisms of the dilator action of Danshen (Salvia miltiorrhiza) on rat isolated femoral artery

This study investigates the actions of Danshen crude extract (Salvia miltiorrhiza) on rat isolated femoral artery rings precontracted with phenylephrine. Low concentrations of Danshen (10 to 30 microg/mL) enhanced the phenylephrine-precontracted tone by a maximum of 31.20+/-2.71%. At concentrations 100 microg/mL or above, Danshen relaxed the precontracted tone, with full relaxation obtained at 1 mg/mL. Involvement of endothelium-dependant mechanisms in the dilator effect of Danshen was investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 microM), a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on the Danshen-induced effect. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a histamine H2 receptor antagonist cimetidine (10 microM), a beta-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 microM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), and a potassium channel inhibitor tetraethylammonium (TEA, 10 and 100 mM); only TEA was effective in partially inhibiting the Danshen-induced effect. These findings suggest the dilator action of Danshen on rat femoral artery was mediated in part by the opening of TEA-sensitive K+ channels in the smooth muscle cells. Muscarinic receptors, histamine receptors, beta-adrenoceptors, endothelium-derived relaxant factors, adenylyl cyclase, and guanylyl cyclase-dependent pathways did not play a role in its vasodilatory effect.     

Sivelestat relaxes porcine coronary artery via inhibition of Ca2+ sensitization induced by a receptor agonist

Sivelestat sodium hydrate (sivelestat) is a novel synthetic drug and specific inhibitor of neutrophil elastase that has been approved in Japan as a treatment for acute lung injury associated with systemic inflammatory response syndrome. There are no reports on the effects of sivelestat on the contractile regulation of vascular smooth muscle. The purpose of the present study was to assess the effects of sivelestat on porcine coronary artery. Sivelestat induced concentration-dependent (3 x 10 to 3 x 10 M) vasorelaxation in U46619 (100 nM)-precontracted porcine coronary artery with or without endothelium. Simultaneous measurements of tension and the cytosolic Ca concentration ([Ca]i) revealed that sivelestat shifted the [Ca]i-tension curve to the right and downward during stimulation with 118 mM K and 100 nM U46619. In beta-escin-permeabilized arterial strips, sivelestat abolished GTP plus U46619-induced contractions at constant [Ca]i, whereas it had no effect on Ca-induced contractions. Thus, sivelestat relaxes porcine coronary artery smooth muscle via the selective inhibition of Ca sensitization induced by a receptor agonist, without affecting Ca-induced contraction.     

GABA(B), opioid and alpha2 receptor inhibition of calcium channels in acutely-dissociated locus coeruleus neurones.

1. The effects of GABA(B), opioid and alpha2 receptor activation on different subtypes of calcium channels in acutely-dissociated rat locus coeruleus (LC) neurones were investigated using whole-cell patch clamping. 2. Barium currents through calcium channels could be fractionated into four classes: L-type (nimodipine-sensitive), N-type (omega-conotoxin GVIA-sensitive), P/Q-type (omega-agatoxin IVA-sensitive) and R-type (remaining in the presence of all three blockers). The percentage of each was, respectively, 25+/-2, 34+/-1, 28+/-3 and 12+/-1% (mean+/-s.e.mean, n=4). 3. The GABA(B) receptor agonist, baclofen, and the opioid receptor agonist, enkephalin, partially inhibited the total barium current in a concentration-dependent manner with EC50 values of 2 and 0.3 microm , respectively. Maximal inhibition was 17+/-1% (n=38) for baclofen and 30+/-2% (n=20) for enkephalin. The alpha2-adrenoceptor agonist, UK14304 (10 microM), also inhibited barium current in these neurones (28+/-2%, n=11). The agonists did not shift the current-voltage relationship along the voltage axis. 4. Maximal baclofen inhibition of different calcium channel subtypes was 9+/-7% (L-type, n=4), 11+/-8% (N-type, n=4), 26+/-6% (P/Q-type, n=4), and 6+/-5% (R-type, n=5). The corresponding values for enkephalin inhibition were 5+/-9% (L-type), 30+/-11% (N-type), 37+/-9% (P/Q-type), and 17+/-8% (R-type). 5. In the presence of a saturating concentration of enkephalin, baclofen produced additional inhibition of the barium current. In contrast, in the presence of a saturating concentration of enkephalin, UK14304 produced no further inhibition of the barium current. 6. These results indicate that neuromodulation of calcium channels in LC neurones involves a complex pattern of overlapping and distinct second messenger pathways. Regulation of LC neuronal firing activity by the modulation of calcium channels may be important for LC-mediated behaviour such as alertness and vigilance.     

Mechanism of inhibition of calcium channels in rat nucleus tractus solitarius by neurotransmitters.

1. High-threshold Ca2+ channel currents were measured every 15 s following a 200 ms voltage step from -80 mV to 0 mV in order to study the coupling mechanism between neurotransmitter receptors and Ca2+ channels in neurones acutely isolated from the nucleus tractus solitarius (NTS) of the rat. 2. Application of 30 microM baclofen (GABAB receptor agonist) caused 38.9 +/- 1.2% inhibition of the peak inward Ba2+ current (IBa2+) in most NTS cells tested (n = 85 of 88). Somatostatin, 300 nM, also reduced IBa2+ by 31.3 +/- 1.6% in 53 cells of 82 tested. 3. Activation of mu-opioid-, GABAB- or somatostatin-receptors inhibited both N- and P/Q-type Ca2+ channels. 4. The inhibition of Ca2+ currents by DAMGo (mu-opioid receptor agonist), baclofen and somatostatin was reduced by treatment with pertussis toxin and partially relieved by application of a 50 ms conditioning prepulse to +80 mV. This suggests that a pertussis toxin-sensitive G-protein was involved in the neurotransmitter-mediated action in the observed inhibition of Ca2+ currents. 5. Intracellular loading with an antiserum raised against the amino terminus of Go alpha (GC/2) markedly attenuated the somatostatin-induced inhibition, but did not block the DAMGO- and baclofen-induced inhibition. 6. These findings suggest at least two different pertussis toxin-sensitive G-protein-mediated pathways are involved in receptor-induced inhibition of Ca2+ currents in the NTS.     

Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine metabolism via the trans-sulphuration pathway in rats

Background and purpose:

Elevated plasma total homocysteine (tHcy) level has been established as an independent risk factor for cardiovascular diseases. Danshensu, an active ingredient of Salvia miltiorrhiza, shows wide cardiovascular benefit. However, in terms of its own methylation, danshensu could elevate tHcy level, which would act against its cardiovascular benefit, thus posing a ‘therapeutic paradox’. As this paradox has not been fully assessed, we have evaluated the effects of danshensu on tHcy levels to uncover the underlying mechanisms.

Experiment approach:

We evaluated the influence of danshensu on homocysteine (Hcy) metabolism in rats with normal tHcy levels and in rat models of elevated tHcy (single intravenous methionine loading model and a hyperhomocysteinemic model after 3 weeks methionine dosing, with and without 3 weeks of danshensu treatment). We also quantified some metabolic intermediates (S-adenosyl methionine, S-adenosyl-l-homocysteine, cysteine and glutathione) relevant to Hcy metabolism in rat liver and kidney.

Key results:

Acute treatment with a single dose of danshensu in rats with normal tHcy did not change plasma tHcy. In contrast, danshensu significantly lowered tHcy in rats with elevated tHcy. The relatively higher cysteine and glutathione levels after treatment with danshensu indicated that its tHcy-lowering effect was via increased activity of the trans-sulphuration pathway.

Conclusions and implications:

Our results suggested that danshensu may act both acutely to increase trans-sulphuration and after chronic exposure to up-regulate the activity of the trans-sulphuration enzymes. The tHcy-lowering effect of danshensu is another cardiovascular benefit provided by S. miltiorrhiza and suggests a potential tHcy-lowering therapy.     

Biological activities of salvianolic acid B from Salvia miltiorrhiza on type 2 diabetes induced by high-fat diet and streptozotocin

Abstract

Context: Salvia miltiorrhiza Bge. (Labiatae) has been widely used for treating diabetes for centuries. Salvianolic acid B (SalB) is the main bioactive component in Salvia miltiorrhiza; however, its antidiabetic activity and possible mechanism are not yet clear.

Objective: To investigate the effects of SalB on glycometabolism, lipid metabolism, insulin resistance, oxidative stress, and glycogen synthesis in type 2 diabetic rat model.

Materials and methods: High-fat diet (HFD) and streptozotocin-induced diabetic rats were randomly divided into model group, SalB subgroups (50, 100, and 200?mg/kg), and rosiglitazone group.

Results: Compared with the model group, SalB (100 and 200?mg/kg) significantly decreased blood glucose (by 23.8 and 21.7%; p?<?0.05 and p?<?0.01) and insulin (by 31.3 and 26.6%; p?<?0.05), and increased insulin sensitivity index (by 10.9 and 9.3%; p?<?0.05). They also significantly decreased total cholesterol (by 24.9 and 27.9%; p?<?0.01), low-density lipoprotein cholesterol (by 56.2 and 64.6%; p?<?0.01), non-esterified fatty acids (by 32.1 and 37.9%; p?<?0.01), hepatic glycogen (by 41.3 and 60.5%; p?<?0.01), and muscle glycogen (by 33.2 and 38.6%; p?<?0.05), and increased high-density lipoprotein cholesterol (by 50.0 and 61.4%; p?<?0.05 and p?<?0.01), which were originally altered by HFD and streptozotocin. In addition, SalB (200?mg/kg) markedly decreased triglyceride and malondialdehyde (by 31.5 and 29.0%; p?<?0.05 and p?<?0.01), and increased superoxide dismutase (by 56.6%; p?<?0.01), which were originally altered by HFD and streptozotocin.

Discussion and conclusion: The results indicate that SalB can inhibit symptoms of diabetes mellitus in rats and these effects may partially be correlated with its insulin sensitivity, glycogen synthesis and antioxidant activities.     

Rapid inhibition of the contraction of rat tail artery by progesterone is mediated by inhibition of calcium currents

Progesterone induced rapid relaxation of KCl-contracted tail artery helical strips from rats. The effect was dose dependent, with an IC50 (inhibitory concentration which produces 50% of the maximal response) of 8.9 microM progesterone. The actions of progesterone were not blocked by bicuculline, indicating that in this tissue the non-genomic actions of progesterone were not mediated via a gamma-aminobutyric acid (GABA)-A receptor. Fura-2 was used to measure intracellular calcium levels ([Ca(2+)](i)) in isolated vascular smooth muscle cells (VSMC). Incubation of cultured VSMC for 15 min with progesterone (10 microM) resulted in an inhibition of the KCl-induced [Ca(2+)](i )increase. The whole-cell patch-clamp technique was used to examine Ca(2+)-channel currents in the membrane of isolated VSMC. Progesterone suppressed the L-type Ca(2+)-channel currents in cells held at a potential of -40 mV. The effects of progesterone were quickly reversed by washout in all three experimental protocols suggesting that these effects on vascular tissues are non-genomic. The correlation of the effects on all these preparations, their time course and reversibility suggested that the rapid relaxation of the rat tail artery induced by progesterone is mediated at least in part by inhibition of L-type calcium channels, leading to inhibition of calcium responses in the VSMC of this tissue.     

Vasorelaxant effect of isopropyl 3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoate, a novel metabolite from Salvia miltiorrhiza, on isolated rat mesenteric artery

The present study was designed to investigate the relaxant effect of isopropyl 3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a new metabolite from Salvia miltiorrhiza, on rat mesenteric artery. Isolated mesenteric arterial rings were mounted in organ baths and the isometric tension changes were measured continuously by a sensitive myograph system. The results showed that IDHP at concentrations greater than 0.1 nM produced a concentration-dependent relaxation of artery contracted by norepinephrine with pEC(50) of 7.41+/-0.08. Removal of the endothelium did not affect this relaxation, suggesting that IDHP exerted a direct effect on vascular smooth muscle cells. Meanwhile, the vasorelaxant effect of IDHP was unaffected by pre-treatment with ATP-sensitive K(+) channel inhibitor glibenclamide, delayed rectifier K(+) channel inhibitor 4-aminopyridine, inwardly rectifying K(+) channel inhibitor barium chloride and beta-adrenoceptor antagonist propranolol. However, the non-specific K(+) channel inhibitor tetraethylammonium (TEA, 3 mM) produced a rightward shift of 1.8 fold on the concentration-response curve of IDHP. Moreover, IDHP shifted the concentration-response curve of CaCl(2) as well as two receptor-mediated constrictors, phenylephrine and 5-hydroxytryptamine, to the right in a non-parallel manner. In the absence of extracellular Ca(2+), IDHP depressed the contractions induced by norepinephrine and CaCl(2), and the maximal inhibitions were 48.3+/-18.9% and 58.4+/-10.9%, respectively. These results suggest that IDHP exerts a vasorelaxant effect by inhibiting both Ca(2+) release from intracellular stores and Ca(2+) influx through voltage-dependent calcium channels, and receptor-operated calcium channels in vascular smooth muscle cells. In addition, activation of vascular TEA-sensitive K(+) channels may be partially involved in the relaxant effect of IDHP.     

Selective modulation by membrane potential of the interaction of some calcium entry blockers with calcium channels in rat mesenteric artery.

1. The effects of flunarizine, (+)-PN 200-110 and nifedipine on [3H]-(+)-PN 200-110 specific binding were investigated in intact rat mesenteric arteries bathed in physiological solution or in KCl-depolarizing solution, and in a membrane fraction from rat mesenteric arteries. 2. Unlabelled dihydropyridines, (+)-PN 200-110 and nifedipine, inhibited [3H]-(+)-PN 200-110 specific binding concentration-dependently in polarized as well as in depolarized intact arteries. The Ki value of (+)-PN 200-110 was decreased in arteries bathed in KCl-depolarizing solution compared to arteries bathed in physiological solution, while the Ki value of nifedipine was not significantly changed. Ki values measured in depolarized arteries were close to the IC50 values (concentrations inhibiting by 50% the KCl-contraction of rat mesenteric artery). 3. Flunarizine (10(-6) M) was unable to displace the specific binding of [3H]-(+)-PN 200-110 in intact arteries bathed in physiological solution. At 10(-7) M-10(-6) M, it inhibited the binding in depolarized arteries, suggesting that prolonged depolarization is required for the interaction of flunarizine with the dihydropyridine receptor. 4. In a membrane fraction isolated from rat mesenteric arteries, (+)-PN 200-110, nifedipine and flunarizine were all able to displace completely the specific binding of [3H]-(+)-PN 200-110. Displacement curves were parallel and Hill coefficients were close to unity. Ki values were close to the values obtained in depolarized intact arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

二十二碳六稀酸对大鼠冠状动脉平滑肌细胞BKCa单通道的作用

目的 探讨二十二碳六烯酸(DHA)对大鼠冠状动脉平滑肌细胞(CASMCs)大电导钙激活性钾通道(BKca)的影响.方法 采用酶消化法获得大鼠CASMCs,用膜片钳技术以单通道内膜向外模式记录在跨膜电位60 mV下,加入0、10、20、30、40、50、60、70和80μmol/L DHA后,大鼠CASNXs的BKca单通道开放概率(Po)、电流幅度值(Am)、平均开放时间(To)、平均关闭时间(Tc)的变化.结果 在跨膜电位60mV时,当DHA浓度＞10/μmol/L时,BKca通道的Po增大,且呈浓度依赖性,Po从(0.072±0.003)(0 μmol/L DHA,n=6)增至(0.606±0.089)(80μmol/L DHA,μ=10)(P＜0.05),EC50是(36.11±0.08)μmol/L,当DHA浓度＜10μmol/L时,Po增加不明显;加入不同浓度的DHA时,对通道Am及To的作用不明显,通道Tc明显缩短,Tc由(492.91±42.12)ms(0μmol/L DHA,n=6)缩短至(72.39±6.97)ms(80μmol/L DHA,n=10)(P＜0.05).结论 DHA能直接激活BKca通道而产生舒张血管、增加血流量的效应.     

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.