Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro

Aim： Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase （MLCK） and Rho kinase （ROCK）. The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro.
Methods： Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers.

Results： Addition of carbachol （0.4-4 μmol/L） into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine （1 μmol/L）. Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 （each at 10 μmol/L） significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme （either MLCK or ROCK） was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients （r=0.52, P〈0.05）.

Conclusion： Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity.     

The involvement of protein kinase C and tyrosine kinase in vanadate-induced contraction

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl₃ and significantly inhibited by 10 μM verapamil and 1 μM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca²⁺ concentration, and the influx of extracellular Ca²⁺ was mediated through voltage-dependent Ca²⁺ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 μM) and sodium nitroprusside (1 μM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca²⁺ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.     

Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein

BACKGROUND AND PURPOSE: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM-254890 (YM), a Gq/11-specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh). EXPERIMENTAL APPROACH: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of M3 receptors and the alpha subunit of Gq/11 (Galpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. KEY RESULTS: Both phasic and tonic components of contractions evoked by 2 microM CCh were inhibited by YM (3-10 microM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 microM) evoked an NSCC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 microM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M3 receptors and Galpha(q/11). CONCLUSIONS AND IMPLICATIONS: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G(q/11)-coupled pathway. YM is a useful tool to assess whether Gq/11 is involved in a signal transduction system.     

Potentiation of beta-adrenoceptor function in bovine tracheal smooth muscle by inhibition of protein kinase C

To examine the role of contractile agonist-induced activation of protein kinase C (PKC) in functional antagonism of airway smooth muscle contraction by beta-adrenoceptor agonists, we examined the effects of the specific PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide) on isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by various concentrations of methacholine and histamine. In the absence of GF 109203X, the potency of isoprenaline (pD(2)) was gradually reduced at increasing methacholine- and histamine-induced smooth muscle tones, but the maximal relaxation (E(max)) was decreased only at higher concentrations of methacholine. In the presence of GF 109203X, pD(2) values were significantly increased for both methacholine- and histamine-induced contractions. Moreover, isoprenaline E(max) values in the presence of high concentrations of methacholine were also increased. Although both methacholine- and histamine-induced contractions were slightly reduced by GF 109203X, the changes in isoprenaline pD(2) could only partially be explained by reduced contractile tone. In contrast to isoprenaline, forskolin-induced relaxations were not affected by GF 109203X. The results indicate that PKC activation contributes to the reduced beta-adrenergic responsiveness induced by methacholine and histamine, which may involve uncoupling of the beta-adrenoceptor from the effector system. Since many mediators and neurotransmitters in allergic airway inflammation can activate PKC, this cross talk may be important in the reduced bronchodilator response of patients with severe asthma.     

拉马克啉对内皮素-1诱导的大鼠血管平滑肌细胞增殖及蛋白激酶C表达的作用

目的：探讨拉马克啉（levcromakalim）对内皮素-1（ET-1）诱导的大鼠血管平滑肌细胞增殖及蛋白激酶C（protein kinase C，PKC）表达的影响。方法：体外培养Wistar大鼠主动脉血管平滑肌细胞（vascular smooth muscle cell，VSMCs），用ET-1诱导其增殖，用不同浓度拉马克啉共培养，MTT法评价VSMCs增殖情况，3H—TdR检测DNA合成，流式细胞术检测VSMCs凋亡，逆转录聚合酶链式反应（RT-PCR），Western blot检测PKCamRNA及蛋白表达。结果：拉马克啉对ET-1所致VSMCs增殖有显著抑制作用，随浓度增加，其MTT活性细胞含量和3H—TdR掺入量都明显减少（P〈0．05）；拉马克啉呈剂量依赖性地增加G0／G1期VSMCs（P〈0．05），促使VSMCs凋亡增多（P〈0．05）；拉马克啉抑制VSMCs内PKCamRNA及蛋白表达。结论：拉马克啉抑制ET-1诱导的VSMCs增殖作用，可能的机制是通过下调VSMCs内PKCa表达水平而影响vSMCs增殖和凋亡。     

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats

Background and purpose:

Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca²⁺ sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca²⁺ sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity.

Experimental approach:

Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca²⁺ concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries.

Key results:

Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca²⁺ sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers.

Conclusions and implications:

We conclude that in DM enhanced myofilament Ca²⁺ sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.     

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

In cultured canine tracheal smooth muscle cells (TSMCs), muscarinic receptor stimulation led to phosphoinositide (PI) hydrolysis. formation of inositol phosphates (IPs), and mobilization of intracellular Ca²⁺. Desensitization of IPs accumulation and Ca²⁺ mobilization evoked by carbachol was investigated using [³H]inositol labelling and Ca²⁺-sensitive dye fura-2. Treatment of TSMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min blocked the carbachol-stimulated formation of IPs and mobilization of Ca²⁺. The concentrations of PMA that gave half-maximal and maximal inhibition of carbachol-induced IPs accumulation were 70 nM and 1 M. respectively. The inhibitory effect of PMA on carbachol-induced responses was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Treatment of TSMCs with PMA for 24 h, the cells remained the ability to response to carbachol-induced IPs accumulation and Ca²⁺ mobilization with the same extent as that observed in the control group. Inactive phorbol ester, 4-phorbol 12, 13-didecanoate at 1 M, did not inhibit the responses. The K_D and B_max of the muscarinic receptor for [³H]N-methyl scopolamine binding were not significantly changed by PMA treatment for either 30 min or 24 h. The locus of this inhibition was further investigated by examining the effect of PMA on AIF _inf4 ^sup– -stimulated IPs accumulation in canine TSMCs. AIF _inf4 ^sup– -induced response was inhibited by PMA treatment. supporting that G protein(s) can be directly activated by AIF _inf4 ^sup– which was uncoupled to phospholipase C (PLC) by PMA treatment. The concomitant loss of IPs and Ca²⁺ mobilization is strong evidence in support of a causal relationship between PKC and IPs or Ca²⁺ pathways. In addition. our findings suggest that activation of PKC leads to a negative feedback regulation of carbachol-induced responses at a level distal to receptor occupancy. Correspondence to: Mao Yang at the above address     

马齿苋总黄酮对缺血缺氧刺激下血管平滑肌细胞蛋白激酶C活性的影响

目的观测马齿苋总黄酮(PTF)对缺血缺氧刺激下血管平滑肌细胞钙超载及蛋白激酶C(PKC)的影响。方法用Fura-2/AM作Ca2+指示剂,检测PTF对正常培养及缺血缺氧作用下家兔主动脉血管平滑肌细胞Ca2+浓度及PKC的活性的改变。结果 PTF对正常培养家兔主动脉血管平滑肌细胞[Ca2+]i浓度无明显影响;PTF(8,16,32,64 mg·L-1)剂量依赖性抑制缺血缺氧缺氧致血管平滑肌细胞[Ca2+]i升高;PTF对正常培养家兔主动脉血管平滑肌细胞胞浆、胞膜PKC活性均升高;PTF处理后胞浆PKC活性下降,胞膜PKC活性上升。结论PTF可能抑制缺氧致血管平滑肌细胞钙超载,调节缺氧条件下血管平滑肌细胞PKC活性。     

The minor population of M3‐receptors mediate contraction of human detrusor muscle in vitro

1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro. 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4‐diphenylacetoxy‐N‐methyl piperidine methiodide (4‐DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pK_B values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M₁, M₂ and M₄‐receptors, respectively, all had low affinities on the human detrusor (pK_B values of 6.8, 6.9 and 6.5, respectively), whilst the M₃‐selective antagonist 4‐DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3‐ and m5‐muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M₃‐muscarinic receptor subtype with no contribution from the major M₂‐receptor population.     

Effects of 17β-estradiol and progesterone on mitogen-activated protein kinase expression and activity in rat uterine smooth muscle

Activation of mitogen-activated protein kinases (MAPKs) is a critical event in mitogenic signal transduction. MAPKs are activated by tyrosine phosphorylation and translocate to different cellular compartments affecting protein function and gene expression. MAPK expression and activity was examined in uterine smooth muscle from rats pretreated with estradiol-17β alone or with estradiol-17β and progesterone. MAPK expression was detected by immunoblotting using erk 1/2 antibodies. MAPK activity was detected by measurement of the phosphorylation of a MAPK-specific peptide sequence of myelin basic protein. Steroid treatment caused a modest (20%) decline in erk 1 and 2 expression in membrane and cytosolic fractions. Both estrogen and progesterone increased MAPK tyrosine phosphorylation and membrane-associated MAPK activity. Steroid treatment increased cytosolic MAPK tyrosine phosphorylation, but not enzymatic activity. These data suggest that gonadal steroid hormones, which stimulate uterine hypertrophy, may exert their hypertrophic effects by increasing MAPK activity.     

Endothelin-1 induces contraction via a Syk-mediated p38 mitogen-activated protein kinase pathway in rat aortic smooth muscle

Although spleen tyrosine kinase (Syk) has crucial roles in various cells, its function on vascular smooth muscle contraction has not been determined. In the present study, we performed experiments to determine if Syk contributes to the endothelin-1 (ET-1)-mediated contraction in rat aortic smooth muscle. ET-1-induced contraction of aortic strips was inhibited by piceatannol, PD98059, and SB203580, inhibitors of Syk, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK), respectively. Piceatannol also attenuated high K(+)-induced contraction. ET-1 dose-dependently enhanced the activity of Syk and this was inhibited by piceatannol in both rat aortic strip and rat aortic smooth muscle cells. The phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), but not that of ERK1/2, in response to ET-1 was inhibited by both piceatannol and SB203580. These results suggest that Syk may play an important role in the regulation of aortic smooth muscle contraction induced by ET-1, which may be mediated by the p38 MAPK/HSP27 signaling pathway.     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

Effect of 2,3-butanedione monoxime on force of contraction and protein phosphorylation in bovine smooth muscle

The aim of the study was to investigate the effects of the putative protein phosphatase (PP) activator 2,3-butanedione monoxime (BDM) in vascular smooth muscle. BDM concentration-dependently increased PP activity in homogenates of bovine coronary arteries and led to dephosphorylation of various smooth muscle proteins in ³²P-labelled bovine aortic smooth muscle cells. In isolated bovine coronary artery rings (CARs) the effects of 10 mmol/l BDM on force of contraction (FOC) under conditions of depolarization by 75 mmol/l KCl and PP inhibition by 100 μmol/l cantharidin were investigated. At the end of contraction experiments CARs were freeze-clamped and myosin light chain (MLC₂₀) phosphorylation was determined by two-dimensional gel electrophoresis. Pretreatment of CARs with BDM reduced KCl-induced FOC to 42 ± 4% vs. 118 ± 1% (no BDM) and cantharidin-induced FOC to 102 ± 2% vs. 120 ± 7% (no BDM) compared to a former KCl contraction (= 100%). Moreover, BDM increased the amount of unphosphorylated MLC₂₀ up to 56 ± 2% vs. 36 ± 5% (no BDM) and 28 ± 2% vs. 21 ± 1% (no BDM), respectively, demonstrating the central role of MLC₂₀ phosphorylation in initiating smooth muscle contraction. In KCl precontracted CARs BDM decreased FOC to 47 ± 4% vs. 100 ± 1% (no BDM) but did not affect MLC₂₀ phosphorylation, suggesting an uncoupling of force maintenance and MLC₂₀ phosphorylation. In contrast, BDM neither affected FOC nor MLC₂₀ phosphorylation in CARs precontracted with cantharidin. These results strengthen the hypothesis that PP activation by BDM only occurs on the holoenzyme level, e.g. by affecting regulatory subunits. Received: 15 July 1998 / Accepted: 22 March 1999     

蛋白激酶C与血管平滑肌α_1肾上腺素受体触发Ca~（2＋）内流的关系

在酶新鲜分离的犬肠系膜上动脉平滑肌细胞，蛋白激酶Ｃ（ＰＫＣ）的激活剂佛波二丁酯（ＰＤＢ）引起的胞内Ｃａ２＋增高作用可被ＰＫＣ抑制剂１－（５－异喹啉磺酰）－２－甲基哌嗪（Ｈ７）所阻断，而哌唑嗪和普萘洛尔则不能阻断ＰＤＢ的这一作用；１０μｍｏｌ·Ｌ－１苯福林引起的胞内Ｃａ２＋增高作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７部分阻断；在无Ｃａ２＋液，Ｈ７可部分抑制苯福林引起的内Ｃａ２＋释放和外Ｃａ２＋内流，两者分别被抑制了３３±３％和５８±６％；ＫＣｌ（２０－１００ｍｍｏｌ·Ｌ－１）可浓度依赖性地引起胞内钙升高，这一作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７不同程度地阻断；ＰＤＢ引起的胞内Ｃａ２＋增高也可分别被１．２５和２．５μｍｏｌ·Ｌ－１维拉帕米部分和全部阻断。上述结果提示ＰＫＣ参与苯福林引起的部分内Ｃａ２＋释放和外Ｃａ２＋内流，但以参与外Ｃａ２＋内流为主；这一作用可能与ＰＫＣ激活，引起电压依赖性Ｃａ２＋通道开放有关。     

Influence of protein kinase C-stimulation by a phorbol ester on neurotransmitter release at frog end-plates

Summary 1. The effect of the phorbol ester 12-O-tetradecanoylphorpbol-13-acetate (TPA) on the stimulation-evoked neurotransmitter release has been investigated by measuring the quantal content (m) of end-plate potentials at frog neuromuscular junctions (Rana temporaria, M. sartorius). 2. After addition of TPA (0.1 up to 1 mol/1) to the Ringer solution the m-values increased in a concentration-dependent manner up to more than 3 times the control values. 3. Inhibition of the activity of the protein kinase C through the inhibitor 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) blocked this effect of TPA. 4. The TPA effect was much more conspicuous when the m-value was reduced by raising the extracellular Mg²⁺ concentration. Between the control m-values and the n-fold increase in the m-value enhanced by TPA a hyperbolic relation was observed. 5. It is concluded that protein kinase C stimulation affects predominantly the spontaneous release of neurotransmitter at the frog neuromuscular junction and only very poorly the stimulation-evoked one. Send offprint requests to C. G. Caratsch     

己酮可可碱与蛋白激酶C抑制剂对佛波醇酯诱导脑微血管内皮细胞表达细胞间粘附分子-1的影响

目的:研究佛波醇酯(PMA)诱导大鼠脑微血管内皮细胞(RBMEC)表达细胞间粘附分子-1(ICAM-1)及PKC抑制剂H7与己酮可可碱(PTX)的抑制作用。方法:采用ELISA方法测定培养RBMEC表达ICAM-1.结果:PMA在10-100nmol·L~(-1)范围内剂量依赖性地诱导RBMEC表达ICAM-1;在4-16h范围内时间依赖性诱导RBMEC表达ICAM-1。H7和PTX分别在5-50μmol·L~(-1)和1-100μmol·L~(-1)范围内剂量依赖性抑制PMA诱导的RBMEC表达ICAM-1。PTX 100μmol·L~(-1),H7 50μmol·L~(-1)时,抑制作用达最大[吸光度分别从(0.410±0.014)降至(0.175±0.022)和(0.182±0.013),P<0.01]。结论:PKC抑制剂及己酮可可碱能抑制PMA诱导RBMEC表达ICAM-1,表明PKC参与RBMEC ICAM-1表达调控。     

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis

The exact mechanisms underlying the weak bronchodilator effect of K_ATP channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 μM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1–100 μM, cumulatively) and HA 1077 (0.01–30 μM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 μM) and chelerythrine (10 μM) increased the pinacidil-induced relaxations by ∼%100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure. Two authors made an equal contribution to this study.     

Phorbol ester-induced contraction through p38 mitogen-activated protein kinase is diminished in aortas from DOCA-salt hypertensive rats

The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in Ca2+-free medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from sham-operated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the Ca2+-independent contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.     

虎杖苷对缺血缺氧下平滑肌细胞蛋白激酶C的影响

目的　观察虎杖苷对缺血缺氧作用下血管平滑肌细胞(VSMC)蛋白激酶C(PKC)活性的影响以探讨其抗休克作用的机制。方法　取大鼠胸主动脉培养VSMC,用Koyama方法复制VSMC缺血缺氧模型。用液体闪烁计数仪测定PKC的活性。结果　缺血缺氧状态下VSMC胞浆PKC的活性上升(vs正常组P<0 .05),但胞膜的PKC活性下降(vs正常组P<0 .05),经PD治疗后胞浆PKC的活性下降(vs缺血缺氧组、治疗对照组P<0. 05,vs正常组P>0 05 ),胞膜的PKC活性上升(vs缺血缺氧组、治疗对照组P<0 .05,vs正常组P>0 .05)。结论　PD对缺血缺氧作用下的VSMCPKC的活性有调节作用,这可能是PD抗休克作用的分子机制之一。