硝酸甘油耐受性机制探讨及卡维地洛的预防作用

目的　研究硝酸甘油 (NTG)连续应用耐受性的形成机制及卡维地洛对 NTG耐受性的预防作用。方法　大鼠 NTG 10 mg· kg- 1 皮下注射 ,每日 3次连用 6日引致耐受。测定耐受性形成前、后舌静脉注射 NTG对鼠尾容积的作用 ,作为硝酸甘油效应及耐受的指标 ,同时检测耐受性形成后有关组织的氧自由基 (· O2 - )、亚硝酸盐(NO2 - )、总硝酸盐 (NOX- )、超氧化物歧化酶 (SOD)活力和丙二醛 (MDA)。结果　大鼠连用 NTG后 ,再静注 NTG对鼠尾容积的增加作用明显减弱 ;主动脉和心脏组织及血浆中· O2- 和 NOX- 都明显升高 ,除血浆 NO2- 水平升高外其余组织 NO2 - 水平都明显降低 ;主动脉、肝脏和红细胞中的 MDA含量增高 ,SOD活力降低 ;卡维地洛能有效预防NTG连续应用耐受性的形成 ,完全或部分防止上述检测指标的变化。结论　 NTG连续应用期间耐受性的形成与· O2- 水平升高引起一氧化氮 (NO)氧化灭活有关 ;卡维地洛通过抗氧化、清除· O2- 等作用而有效预防或减轻 NTG连用过程中耐受性的形成。     

Loss of preconditioning in rabbits with vascular tolerance to nitroglycerin.

A preceding right ventricular overdrive pacing (VOP) of 500 b.p.m. for 5 min, markedly reduced the severity of global myocardial ischaemia produced by a subsequent 5-min VOP in conscious rabbits. This VOP-induced preconditioning developed in parallel with an increase in cardiac cyclic guanosine 3':5'-monophosphate (cyclic GMP) content. VOP-induced preconditioning was abolished when the animals had been made tolerant to the vasodilator effect of nitroglycerin (NG). In the heart of the NG-tolerant rabbits, neither VOP nor preconditioning increased cyclic GMP content. This suggests that changes by NG tolerance of cyclic GMP metabolism may account for the loss of VOP-induced preconditioning.     

Differential and selective inhibition of protein kinase A and protein kinase C in intact cells by balanol congeners.

The fungal metabolite balanol is a potent inhibitor of protein kinase A (PKA) and protein kinase C (PKC) in vitro that acts by competing with ATP for binding (K(i) approximately 4 nM); congeners of balanol show specificity for PKA over PKC. We have characterized the effects of balanol and 10"-deoxybalanol in intact cells to determine whether these compounds cross the cell membrane and whether the potency and specificity noted in vitro are preserved in vivo. In neonatal rat myocytes and cultured A431 cells transiently transfected with a cyclic AMP response element-luciferase reporter construct, balanol inhibits the induction of luciferase activity by isoproterenol, indicating inhibition of PKA. Western analysis shows that both balanol and 10"-deoxybalanol reduce phosphorylation of cAMP response element-binding protein in isoproterenol-stimulated A431 cells; inhibition is concentration dependent with an IC(50) value of approximately 3 microM. Balanol, but not 10"-deoxybalanol, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, a PKC substrate, in phorbol ester-stimulated A431 cells (IC(50) approximately 7 microM). Our data demonstrate that balanol is a potent inhibitor of PKA and PKC in several whole-cell systems and causes no obvious toxicity. In addition, balanol congeners inhibit PKA and PKC with the specificity and potency predicted by in vitro experiments.     

Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers

P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.     

维生素C对实验兔硝酸甘油耐受性影响的研究

目的探讨维生素C对硝酸甘油耐受性的影响。方法选取30只兔龄5~6个月的健康家兔,将取出的兔胸主动脉环按随机数字表法分为3组:对照组、硝酸甘油组、硝酸甘油+维生素C组,每组10份,分别做硝酸甘油量效关系及超氧化物歧化酶(SOD)活力和丙二醛(MDA)水平测定。结果对照组的最大舒张程度为(96±7)%,硝酸甘油组的最大舒张程度为(63±7)%,硝酸甘油+维生素C组的最大舒张程度为(86±5)%,硝酸甘油组、硝酸甘油+维生素C组可以明显增强兔离体动脉环对硝酸甘油的反应性。硝酸甘油组血管组织中MDA含量明显要高于对照组和硝酸甘油+维生素C组,差异有统计学意义(P<0.01),而对照组和硝酸甘油+维生素C组的MDA含量差异无统计学意义。硝酸甘油组血管组织中SOD活力明显要低于对照组和硝酸甘油+维生素C组,差异有统计学意义(P<0.05),而对照组和硝酸甘油+维生素C组的SOD活力差异无统计学意义。结论维生素C可改善硝酸甘油的耐受性。     

Lithium inhibition of protein kinase C activation-induced serotonin release

The effects of eight serotonin (5-HT) receptor antagonists on the anorectic effect of d-fenfluramine (3.0 mg/kg, IP) were examined in a test of sweet mash consumption, using non-deprived male rats. d-Fenfluramine's effect was attenuated by the mixed 5-HT₁/5-HT₂ receptor antagonists, methiothepin and metergoline; by the 5-HT₂ receptor antagonist ritanserin; and by (±)cyanopindolol, a mixed 5-HT_1A/5-HT_1B receptor antagonist. In contrast, d-fenfluramine's effect was not antagonised by the 5-HT₂ receptor antagonists ketanserin and ICI 169 369; the 5-HT₃ receptor antagonist ICS 205 930; or by xylamidine, a peripheral 5-HT receptor antagonist. In this feeding model, none of the 5-HT antagonists, when tested alone, had any effect to increase palatable food consumption. The pattern of results obtained strongly suggest that central 5-HT₁ receptors play an important role in the mediation of d-fenfluramine-induced anorexia.     

Evidences for prevention of nitroglycerin tolerance by carvedilol.

Carvedilol, a beta-blocker has shown clinically to attenuate the development of nitroglycerin (NTG) tolerance. The present study was designed to investigate the possible mechanisms whereby carvedilol could prevent NTG tolerance, particularly at the level of vascular superoxide anion (O2-) production (an important factor in nitrate tolerance) as well as modulation of certain aortic antioxidants. Rabbits were treated with NTG patch (1.5 microg/kg/min) and/or cavedilol (10 mg/kg/day) for 3 days. Relaxation of aortic segments was studied in organ chamber and rate of vascular O2- production was determined. In addition, aortic glutathione (GSH) level and superoxide dismutase (SOD) activity was also assessed. Aortic segments from NTG-treated rabbits showed a significant decrease in maximal relaxation in response to various vasodilators. Also, NTG treatment increased vascular O2- production by two-fold as compared with untreated control group. The potential source of O2- production was found to be the adventitia. In addition, treatment of rabbits with NTG induced a significant decrease in total GSH level and SOD activity by 46 and 53%, respectively, as compared with the control values. Concomitant treatment of NTG with carvedilol significantly prevented the development of NTG tolerance and normalized the rate of vascular O2- production. Moreover, carvedilol restored the normal level of aortic antioxidants mainly, total GSH and SOD.     

MDL 27,032 relaxes vascular smooth muscle and inhibits protein kinase C.

The smooth muscle relaxant effect of MDL 27,032, 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone, was studied in vitro using strips of femoral arteries and saphenous veins from dogs and trachea from guinea pigs. MDL 27,032 (10(-6)-10(-3) M) produced a concentration-dependent relaxation of arterial and venous strips contracted by carbachol. MDL 27,032 also antagonized contractions of arterial and venous strips produced by phorbol 12,13-dibutyrate (PDB), a protein kinase C activator, both in normal-Ca2+ and zero-Ca2+ medium. The compound inhibited protein kinase C in soluble extracts prepared from saphenous veins of dogs, with an IC50 value of 36.6 microM. MDL 27,032 was more effective against the contractions produced by phenylephrine and serotonin than by KCl in arteries, but no such selectivity was noted in veins. MDL 27,032 (10(-3) M) also inhibited accumulation of inositol phosphates in femoral artery but not in saphenous vein, and this effect may have contributed to the arterial-relaxant effect. Because the vascular smooth muscle relaxant effect of MDL 27,032 was endothelium independent, did not involve blockade of alpha-adrenoceptors or inhibition of cyclic nucleotide phosphodiesterases, stimulation of beta-adrenergic receptors, stimulation of adenosine A2-receptors, or activation of K+ channels, these data suggest that the relaxant effects of MDL 27,032 primarily involve inhibition of protein kinase C.     

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA-induced pressor responses in rats

Background and purpose:

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure.

Experimental approach:

Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis.

Key results:

Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1–1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1–100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies.

Conclusions and implications:

The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.     

Synthesis, cytotoxicity and protein kinase C inhibition of arylpyrrolylmaleimides

A series of novel arylpyrrolylmaleimides was synthesized and evaluated for their in-vitro cytotoxicity against various human cancer cell lines and their protein-kinase C inhibitory activity. Some of the compounds showed high or moderate cytotoxic activity against the tested cell lines. Compound 6b is the most promising compound against the tested cancer cell lines; 6d and 6e showed moderate protein-kinase C inhibition. Structure-activity relationships are discussed based on the experimental data obtained.     

Acute functional tolerance to ethanol mediated by protein kinase Cepsilon.

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCepsilon) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCepsilon (-/-) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCepsilon and resistance of GABA(A) receptors to ethanol. In contrast, in PKCepsilon (-/-) mice, GABA(A) receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCepsilon (-/-) and PKCepsilon (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCepsilon signaling pathway that contributes to GABA(A) receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCepsilon and does not alter PKCepsilon modulation of ethanol preference.     

Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions

BACKGROUND AND PURPOSE: Previous studies have identified the natural polyphenol curcumin as a protein kinase C (PKC) inhibitor. In contrast, we found significant stimulation of PKC activity following curcumin treatment. Thus, the mechanism of curcumin interaction with PKC was investigated. EXPERIMENTAL APPROACH: We employed phosphorylation assays in the presence of soluble or membrane-bound PKC substrates, followed by SDS-PAGE, autoradiography and phosphorylation intensity measurements. KEY RESULTS: Curcumin inhibited PKC in the absence of membranes whereas stimulation was observed in the presence of membranes. Further analysis indicated that curcumin decreased PKC activity by competition with Ca(2+) stimulation of the kinase, resulting in inhibition of activity at lower Ca(2+) concentrations and stimulation at higher Ca(2+) concentrations. The role of the membrane is likely to be facilitation of Ca(2+)-binding to the kinase, thus relieving the curcumin inhibition observed at limited Ca(2+) concentrations. Curcumin was found to mildly stimulate the catalytic subunit of PKC, which does not require Ca(2+) for activation. In addition, studies on Ca(2+)-independent PKC isoforms as well as another curcumin target (the sarcoplasmic reticulum Ca(2+)-ATPase) confirmed a correlation between Ca(2+) concentration and the curcumin effects. CONCLUSIONS AND IMPLICATIONS: Curcumin competes with Ca(2+) for the regulatory domain of PKC, resulting in a Ca(2+)-dependent dual effect on the kinase. We propose that curcumin interacts with the Ca(2+)-binding domains in target proteins. To our knowledge, this is the first study that defines an interaction domain for curcumin, and provides a rationale for the broad specificity of this polyphenol as a chemopreventive drug.     

The N terminus domain of type VI adenylyl cyclase mediates its inhibition by protein kinase C.

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.     

cAMP-dependent protein kinase regulates inhibition of adenosine transport by ethanol.

Ethanol inhibits adenosine uptake, thereby increasing the concentration of extracellular adenosine. Elevation of extracellular adenosine increases intracellular cAMP concentration via activation of adenosine A2 receptors. Extracellular adenosine is also required for the subsequent development of ethanol-induced heterologous desensitization. Here we report that activation of cAMP-dependent protein kinase is necessary for inhibition of adenosine uptake by ethanol and for the consequent accumulation of extracellular adenosine. Ethanol does not inhibit adenosine uptake in mutants of the S49 cell line that lack receptor-stimulated cAMP production (unc cells) or cAMP-dependent protein kinase activity (kin- cells). Forskolin, which bypasses the receptor-coupling defect in unc cells to increase cAMP levels, restores inhibition of adenosine uptake by ethanol. In contrast, in kin- cells forskolin did not restore inhibition of adenosine uptake by ethanol, despite similar increases in cAMP levels. Taken together, these results suggest that cAMP-dependent protein kinase phosphorylates a component of the nucleoside transporter, thereby regulating the sensitivity of adenosine transport to ethanol.     

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.     

The angiotensin II-receptor antagonist losartan does not prevent hemodynamic or vascular tolerance to nitroglycerin.

Tolerance may involve increased production of angiotensin II. We tested the hypothesis that losartan would prevent the development of tolerance to continuous transdermal nitroglycerin (GTN). Twenty volunteers received losartan, 75 mg/day, or placebo in a randomized, double-blind, parallel fashion. After 1 week, continuous transdermal GTN, 0.6 mg/h, was given, in addition to losartan or placebo, to all volunteers for 1 week. Standing systolic blood pressure (SBP) and heart rate were measured, and forearm venous volume responses to sublingual GTN were evaluated. Measurements were made at baseline, after 1 week of losartan versus placebo, 3 h after initial therapy with transdermal GTN, and after 1 week of continuous transdermal GTN given in combination with losartan versus placebo. After sustained GTN therapy, SBP was unchanged from baseline in both groups, indicating that losartan did not prevent the development of tolerance. Tolerance also developed to the forearm venous volume responses and was not prevented by losartan. Therapy with an angiotensin II-receptor antagonist does not prevent the development of tolerance to continuous transdermal GTN.     

Differences in the vascular selectivity and tolerance between the NO donor/beta-blocker PF9404C and nitroglycerin

The vascular selectivity of PF9404C ((2'S),(2S)-3-isopropylamine,1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2'-propanol), a new beta-blocker with nitric oxide (NO)-donor and vasodilator properties, was studied in different rabbit arteries and veins. Phenylephrine (10(-6) M) or 35 mM K+ were used to pre-contract the arteries and veins prior to study the relaxant effects of PF9404C and nitroglycerin. The potency of both drugs to depress the phenylephrine-induced contraction was greater than that shown in the blockade of the K(+)-evoked contraction in most of the vessels studied, with the exception of the central ear artery. PF9404C exhibited about three-fold higher potency than nitroglycerin to relax the majority of the vessels studied, especially when they were contracted with K+, and showed a certain selectivity of action for the renal artery. PF9404C produced autotolerance but this effect was about 20-fold less pronounced than that observed with nitroglycerin. Cross-tolerance in those preparations pre-exposed to PF9404C that were relaxed later on with nitroglycerin was much greater than autotolerance. The tolerance for nitroglycerin was practically abolished in the presence of N-acetylcysteine. However, this was not the case for PF9404C. These results indicate that, although sharing the property of being NO donors, PF9404C and nitroglycerin show a different profile in causing vasodilation; furthermore, the tolerance to this effect is lesser for PF9404C and seems to be mediated by a mechanism different to that of nitrates. This makes PF9404C a nice pharmacological tool to further develop novel NO-donor compounds with a lesser degree of vascular tolerance than those now available.     

Staurosporine-induced neurite outgrowth in PC12 cells is independent of protein kinase C inhibition.

The protein kinase C (PKC) inhibitor staurosporine, a member of the K252a family of fungal alkaloids that are known as protein kinase inhibitors, induces neurite outgrowth in pheochromocytoma PC12 cells. The progressive staurosporine-induced neurotropic effect (EC50 = 50 nM) has the following characteristics: it is evident after 4 hr of incubation, requires the continuous presence of staurosporine, occurs at 37 degrees but not at 4 degrees, and is not blocked by K252a derivatives. Scanning electron micrographs showed long neurites, ruffling, and dense networks in nerve growth factor (NGF)-treated cells and short neurites, flattening, and smooth cell surface in staurosporine-treated cells. [3H]Staurosporine binding, which was time, temperature, and dose dependent, saturated at 5-10 nM. Other kinase inhibitors were poor competitors. The [3H]staurosporine bound over 20 hr at 37 degrees was poorly dissociated by acetic acid wash or unlabeled staurosporine. These results suggest an uptake process occurring at 37 degrees that is required for the neurotropic effect of staurosporine. NGF did not interfere with staurosporine binding, and staurosporine did not affect NGF receptor binding. At neurotropic concentrations of staurosporine, PKC in PC12 cells was completely inhibited. When PKC activity was down-regulated by prolonged exposure to phorbol myristate acetate, PC12 cells responded to staurosporine with neurite outgrowth similar to that of untreated cells. Although the target and mechanism of the neurotropic effects of staurosporine remain to be determined, the observed effects on PKC-deficient cells indicate that PKC may not be required for the neurotropic effect of this compound in PC12 cells. These results suggest that caution should be taken in the interpretation of staurosporine action in vivo, and they provide a pharmacological tool for the development of potential neurotropic drugs.