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1.

Aim:

To explore the mechanisms underlying the protection by SO2 preconditioning against rat myocardial ischemia/reperfusion (I/R) injury.

Methods:

Male Wistar rats underwent 30-min left coronary artery ligation followed by 120-min reperfusion. An SO2 donor (1 μmol/kg) was intravenously injected 10 min before the ischemia, while LY294002 (0.3 mg/kg) was intravenously injected 30 min before the ischemia. Plasma activities of LDH and CK were measured with an automatic enzyme analyzer. Myocardial infarct size was detected using Evans-TTC method. The activities of caspase-3 and -9 in myocardium were assayed using a commercial kit, and the levels of p-Akt, Akt, PI3K and p-PI3K were examined with Western blotting.

Results:

Pretreatment with SO2 significantly reduced the myocardial infarct size and plasma LDH and CK activities, as well as myocardial caspase-3 and -9 activities in the rats. Furthermore, the pretreatment significantly increased the expression levels of myocardial p-Akt and p-PI3K p85. Administration of the PI3K inhibitor LY294002 blocked all the effects induced by SO2 pretreatment.

Conclusion:

The results suggest that the PI3K/Akt pathway mediates the protective effects of SO2 preconditioning against myocardial I/R injury in rats.  相似文献   

2.

Aim:

To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).

Methods:

Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg−1·d−1, ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg−1·d−1, ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.

Results:

Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg−1·d−1) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002.

Conclusion:

Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.  相似文献   

3.
  相似文献   

4.

Aim:

Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.

Methods:

Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.

Results:

The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor LY294002. Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

Conclusion:

LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.  相似文献   

5.

Background and purpose:

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (IKur), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K).

Experimental approach:

hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method.

Key results:

LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC50 of 7.9 µmol·L−1). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k+1) and unbinding (k−1) rate constants were calculated to be 1.6 µmol·L−1−1·s−1 and 5.7 s−1 respectively. Inhibition by LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A.

Conclusions and implications:

LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for LY294002.  相似文献   

6.
Metabotropic glutamate receptor 2/3 (mGluR2/3) agonists were shown previously to nonselectively decrease both cocaine- and food-maintained responding in rats. mGluR2 positive allosteric modulators (PAMs) may represent improved therapeutic compounds because of their modulatory properties and higher selectivity for mGluR2. We analyzed the effects of the selective, brain penetrant, and systemically active mGluR2 PAM potassium 3′-([(2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy]methyl)biphenyl l-4-carboxylate (BINA) and the mGluR2/3 agonist LY379268 on intravenous cocaine self-administration and cocaine-seeking behavior in rats that had short (1 h, ShA) or long (6 h, LgA) access to cocaine. The effects of BINA on food responding and food-seeking behavior were also analyzed. Finally, we examined the effects of BINA on brain reward function and cocaine-induced reward enhancement using the intracranial self-stimulation procedure. BINA decreased cocaine self-administration in both ShA and LgA rats, with no effect on food self-administration. Alternatively, LY379268 nonselectively decreased both cocaine and food self-administration. BINA decreased cue-induced reinstatement of cocaine seeking with no effect on food seeking. The cocaine-induced enhancement of brain reward function was blocked by BINA, although the highest doses of BINA decreased brain reward function when administered alone, suggesting additive, rather than interactive, effects of BINA and cocaine. In conclusion, BINA attenuated the reinforcing and counteracted the reward-enhancing effects of cocaine and decreased cue-induced cocaine-seeking behavior, without affecting behaviors motivated by food reinforcement. The higher selectivity of BINA compared with an mGluR2/3 agonist for drug- vs food-motivated behaviors suggests a therapeutic role for mGluR2 PAMs for the treatment of cocaine addiction and possibly other drugs of abuse.  相似文献   

7.
  1. It has been proposed that protein kinase C (PKC) in sympathetic nerves is activated during action-potential evoked release of noradrenaline and helps maintain transmitter output. We studied this phenomenon further in rat atria radiolabelled with [3H]-noradrenaline.
  2. Noradrenaline release was elevated by continuous electrical stimulation of the atria for 10 min at either 5 or 10 Hz. Two inhibitors of PKC, polymyxin B (21 μM) and Ro 318220 (3 μM), markedly inhibited the release of noradrenaline but only at the higher stimulation frequency.
  3. Further experiments were conducted with 10 Hz stimulation but for shorter train durations. In this case polymyxin B inhibited noradrenaline release during a 10 or 15 s train of impulses but not during a 5 s train. This suggests that PKC effects are induced during the stimulation train by some process.
  4. The diacylglycerol kinase inhibitor R59949 (10 μM), which prevents the breakdown of diacylglycerol, enhanced noradrenaline release elicited by stimulation at 10 Hz for 10 or 15 s. This effect was not seen if polymyxin B was present and suggests that diacylglycerol is the endogenous activator of PKC.
  5. The source of the diacylglycerol may be through phospholipase C pathways, since the phospholipase C inhibitor U73122 (3 μM) inhibited noradrenaline release at 10 Hz for 10 s and the effect was not seen if polymyxin B was also present.
  6. It is unlikely that phospholipase D is the source of diacylglycerol. Although the phospholipase D inhibitor wortmannin (1 μM) inhibited noradrenaline release, this effect was still observed in the presence of polymyxin B. Furthermore ethanol, which inhibits diacylglycerol formation by phospholipase D, had no effect on noradrenaline release.
  7. We therefore suggest that during a train of high frequency pulses phospholipase C is activated and this results in the production of diacylglycerol which in turn activates PKC. This enables the neurones to maintain transmitter release at a high level.
  相似文献   

8.
Ketamine, an NMDA-receptor antagonist, produces cognitive deficits in humans in a battery of tasks involving attention and memory. Nicotine can enhance various indices of cognitive performance, including working memory span capacity measured using the odor span task (OST). This study examined the effects of a sub-chronic ketamine treatment to model cognitive deficits associated with schizophrenia, and to evaluate the effectiveness of nicotine, antipsychotic clozapine, and the novel mGlu2/3 agonist, LY404039, in restoring OST performance. Male hooded Lister rats were trained in the OST, a working memory task involving detection of a novel odor from an increasing number of presented odors until they exhibited asymptotic levels of stable performance. Sub-chronic ketamine exposure (10 and 30 mg/kg i.p. for 5 consecutive days) produced a dose-dependent impairment that was stable beyond 14 days following exposure. In one cohort, administration of graded doses of nicotine (0.025–0.1 mg/kg) acutely restored the performance in ketamine-treated animals, while significant improvements in odor span were observed in control subjects. In a second cohort of rats, acute tests with clozapine (1–10 mg/kg) and LY404039 (0.3–10 mg/kg) failed to reverse ketamine-induced deficits in doses that were observed to impair performance in the control groups. These data suggest that sub-chronic ketamine exposure in the OST presents a valuable method to examine novel treatments to restore cognitive impairments associated with neuropsychiatric disorders such as schizophrenia. Moreover, it highlights a central role for neuronal nicotinic receptors as viable targets for intervention that may be useful adjuncts to the currently prescribed anti-psychotics.  相似文献   

9.
The availability of effective, reliably accessible, and affordable treatments for snakebite envenoming is a critical and long unmet medical need. Recently, small, synthetic toxin-specific inhibitors with oral bioavailability used in conjunction with antivenom have been identified as having the potential to greatly improve outcomes after snakebite. Varespladib, a small, synthetic molecule that broadly and potently inhibits secreted phospholipase A2 (sPLA2s) venom toxins has renewed interest in this class of inhibitors due to its potential utility in the treatment of snakebite envenoming. The development of varespladib and its oral dosage form, varespladib-methyl, has been accelerated by previous clinical development campaigns to treat non-envenoming conditions related to ulcerative colitis, rheumatoid arthritis, asthma, sepsis, and acute coronary syndrome. To date, twenty-nine clinical studies evaluating the safety, pharmacokinetics (PK), and efficacy of varespladib for non-snakebite envenoming conditions have been completed in more than 4600 human subjects, and the drugs were generally well-tolerated and considered safe for use in humans. Since 2016, more than 30 publications describing the structure, function, and efficacy of varespladib have directly addressed its potential for the treatment of snakebite. This review summarizes preclinical findings and outlines the scientific support, the potential limitations, and the next steps in the development of varespladib’s use as a snakebite treatment, which is now in Phase 2 human clinical trials in the United States and India.  相似文献   

10.
  1. Bradykinin has multiple effects on differentiated NG108-15 neuroblastoma×glioma cells: it increases Ins(1,4,5)P3 production and intracellular Ca2+ concentration [Ca2+]i, evokes a Ca2+ activated K+ current (IK(Ca)) and inhibits M current (IM). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects.
  2. Preincubation with 1 or 5 μM U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by 1 μM bradykinin. U73122 10 μM caused total and irreversible inhibition. The inactive analogue U73343 was without effect.
  3. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+]i was increased by 10 μM U73122, but not by U73343. Individual cells responded to 10 μM U73122 with a small increase in [Ca2+]i, followed in some cells by a large further rise.
  4. Pretreatment of whole-cell clamped cells with 1 μM U73122 for 30 min reduced the bradykinin-induced IK(Ca) to a fifth of its normal size. To suppress it totally, a 7–12 min pretreatment with 5 μM U73122 was required. Again, U73343 was without effect.
  5. U73122 and U73343 at concentrations of 5–10 μM irreversibly decreased the holding current (Ih) which at a holding potential of −30 or −20 mV mainly flows through open M channels. The decrease was often preceded by a transient increase.
  6. M current (IM) measured with 1 s pulses, was also decreased by 5–10 μM U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin-induced inhibition of IM was not affected by U73122.
  7. Preincubation with 1 or 3 mM neomycin for 15 min did not affect Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by bradykinin. Pretreatment with 3 mM neomycin for about 20 min diminished the bradykinin-induced IK(Ca) to a fifth of its normal size.
  8. The four main conclusions drawn from the results are: (a) U73122 suppresses bradykinin-induced PLC activation and IK(Ca), but not IM inhibition. (b) This indicates that the transient outward current IK(Ca), but not the decrease of IM in response to bradykinin, is mediated by PLC. (c) U73122 itself inhibits IM and mobilizes Ca2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC-mediated signalling pathways in NG108-15 cells.
  相似文献   

11.
  1. The nonpeptide bradykinin B2 receptor antagonist, FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-(2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide), was tested in models involving bradykinin-induced activation of primary afferent neurones in vitro and in vivo.
  2. Bradykinin-induced contractions of the rabbit isolated iris sphincter muscle mediated by tachykinin release from trigeminal afferent neurones were inhibited in a non-competitive manner by FR173657. A pKB value of 7.9 was calculated. Effects of substance P were unaffected by FR173657.
  3. Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 μmol kg−1 FR173657 s.c. (P<0.05), and completely abolished by 3 μmol kg−1 (P<0.05). Peroral administration of 5 μmol kg−1 FR173657 abolished the bradykinin effects (P<0.05); lower doses had no significant effect.
  4. Shortening by intraplantar injection of bradykinin of the paw withdrawal latency in response to radiant heat was abolished by 3 μmol kg−1 FR173657 s.c. (P<0.05), while 300 nmol kg−1 had an intermediate effect. Hyperalgesia induced by prostaglandin E2 remained unaffected by FR173657.
  5. Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by FR173657 s.c. with an ID50 of 1.1 μmol kg−1, while the peptidic B2 antagonist icatibant (Hoe-140; D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin) caused inhibition at significantly lower doses (ID50 8.5 nmol kg−1 P<0.001). Responses to hydrochloric acid i.p. remained unaffected by FR173657.
  6. FR173657 or similar nonpeptide compounds may be useful for the development of drugs for diseases involving pain induced by the release of endogenous kinins, i.e. especially in acute inflammatory conditions.
  相似文献   

12.
  1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (−) enantiomer R91154 on whole-cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of −80 mV, at 10 s intervals had an average magnitude of 1 nA.
  2. Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration-dependent manner; their IC50s were 1.94 μM for lubeluzole and 2.54 μM for R91154. In a current-voltage protocol, lubeluzole (3 μM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I-V curve were detected.
  3. After 10 min exposure to 3 μM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered.
  4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at −110 mV and 87% at −50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at −110 mV, decreased at −80 mV and did not occur at −50 mV.
  5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 μM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 μM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa.
  6. The effects of lubeluzole (3 μM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At −80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole).
  7. In mouse chromaffin cells approximately half of the whole-cell IBa was sensitive to 3 μM nifedipine (L-type Ca2+ channels) and the other half to 3 μM ω-conotoxin MVIIC (non-L-type Ca2+ channels). In ω-conotoxin MVIIC-treated cells, 3 μM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound.
  8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca2+ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca2+ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca2+ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca2+ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.
  相似文献   

13.
  1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors.
  2. The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 μM) had no effect on basal total inositol phosphate ([3H]-InsPx) accumulation (in the presence of Li+) in myo-[3H]-inositol pre-labelled slices, but enhanced the maximal [3H]-InsPx response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC50 values for DHPG (-log EC50 (M): control, 5.56±0.05; +2R,4R-APDC, 5.51±0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass response over an initial 0–300 s time-course.
  3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [3H]-InsPx accumulation were observed in both the presence and nominal absence of extracellular Ca2+, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(1,4,5)P3 accumulation in the absence or presence of 2R,4R-APDC.
  4. 2R,4R-APDC and (2S, 1′R, 2′R, 3′R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC50 values of 1.3 and 0.04 μM (-log EC 50 (M): 2R,4R-APDC, 5.87±0.09; DCG-IV, 7.38±0.05). In the presence of DHPG (30 μM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [3H]-InsPx accumulation with respective EC50 values of 4.7 and 0.28 μM (-log EC50 (M): 2R,4R-APDC, 5.33±0.04; DCG-IV, 6.55±0.09) which were 3–7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses.
  5. The group II-selective mGlu receptor antagonist LY307452 (30 μM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [3H]-InsPx accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (Kds, 3.7±1.1 and 4.1±0.4 μM respectively) for each response.
  6. The ability of 2R,4R-APDC to enhance receptor-mediated [3H]-InsPx accumulation appeared to be agonist-specific; thus although DHPG (100 μM) and the muscarinic cholinoceptor agonist carbachol (10 μM) stimulated similar [3H]-InsPx accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors.
  7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.
  相似文献   

14.

Aim:

Aliskiren (ALK) is a renin inhibitor that has been used in the treatment of hypertension. The aim of this study was to determine whether ALK could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action.

Methods:

Transverse aortic constriction (TAC) was performed in mice to induce heart pressure overload. ALK (150 mg·kg−1·d−1, po), the autophagy inhibitor 3-methyladenine (10 mg·kg−1 per week, ip) or the PKCβI inhibitor LY333531 (1 mg·kg−1·d-1, po) was administered to the mice for 4 weeks. Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. Mechanically stretched cardiomyocytes of rats were used for in vitro experiments. The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.

Results:

TAC induced marked heart hypertrophy and fibrosis, accompanied by high levels of Ang II in plasma and heart, and by PKCβI/α and ERK1/2 phosphorylation in heart. Meanwhile, TAC induced autophagic responses in heart, i.e. increases in autophagic structures, expression of Atg5 and Atg16 L1 mRNAs and LC3-II and Beclin-1 proteins. These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration. In TAC-mice, 3-methyladenine administration also ameliorated heart hypertrophy, fibrosis and dysfunction, while LY333531 administration inhibited ERK phosphorylation and autophagy in heart. In mechanically stretched cardiomyocytes, CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

Conclusion:

ALK ameliorates heart hypertrophy, fibrosis and dysfunction in the mouse model in setting of chronic pressure overload, via suppressing Ang II-PKCβI-ERK1/2-regulated autophagy.  相似文献   

15.
  1. CP-060S is a novel sodium and calcium overload inhibitor, and is also characterized as a calcium channel blocker. As these activities have each been shown independently to ameliorate ischaemia damage in the myocardium, the combination may synergistically exert cardioprotection. In this study, therefore, the protective effect of CP-060S against ischaemia- and reperfusion-induced arrhythmia was evaluated in anesthetized rats.
  2. Rats were anaesthetized with pentobarbitone, and the left anterior descending coronary artery was occluded for either 5 min with subsequent reperfusion (a reperfusion-induced arrhythmia model) or 30 min without (an ischaemia-induced arrhythmia model). All drugs were intravenously administered 1 min before the onset of occlusion.
  3. In the reperfusion-induced arrhythmia model, the animals in the vehicle-treated group exhibited ventricular tachycardia (VT) in 100%, ventricular fibrillation (VF) in 89%, and death caused by sustained VF in 56%. CP-060S (30–300 μg kg−1) dose-dependently suppressed the incidences of arrhythmias. Significant decreases occurred at 100 μg kg−1 in VF (incidence: 42%) and mortality (8%), and at 300 μg kg−1 in VT (50%), VF (33%) and mortality (8%). This protective effect of CP-060S was 10 times more potent than that of a pure calcium channel blocker, diltiazem (30–1000 μg kg−1) we tested, in terms of effective dose ranges. As both drugs decreased myocardial oxygen consumption estimated by rate-pressure product to a similar extent, the calcium channel blocking activity of CP-060S would not seem to be sufficient to explain its potency.
  4. In the same model, co-administration of ineffective doses of diltiazem (300 μg kg−1) and a sodium and calcium overload inhibitor, R56865 (100 μg kg−1), produced significant suppression of VT (incidence: 62%), VF (46%) and mortality (8%). By contrast, co-administration of R56865 at the same dose with CP-060S (300 μg kg−1) did not add to the effect of a single treatment of CP-060S.
  5. In the ischaemia-induced arrhythmia model, CP-060S (300 μg kg−1) significantly decreased the incidence of VF from 75% to 29%, whereas diltiazem (1 mg kg−1) was ineffective.
  6. These results suggest that CP-060S inhibits both ischaemia- and reperfusion-induced arrhythmia. The combination of the calcium channel blocking effect and the calcium overload inhibition was hypothesized to contribute to these potently protective effects.
  相似文献   

16.

Background and purpose:

Several P2X7 receptor antagonists are allosteric inhibitors and exhibit species difference in potency. Furthermore, N2-(3,4-difluorophenyl)-N1-(2-methyl-5-(1-piperazinylmethyl)phenyl)glycinamide dihydrochloride (GW791343) exhibits negative allosteric effects at the human P2X7 receptor but is a positive allosteric modulator of the rat P2X7 receptor. In this study we have identified several regions of the P2X7 receptor that contribute to the species differences in antagonist effects.

Experimental approach:

Chimeric human-rat P2X7 receptors were constructed with regions of the rat receptor being inserted into the human receptor. Antagonist effects at these receptors were measured in ethidium accumulation and radioligand binding studies.

Key results:

Exchanging regions of the P2X7 receptor close to transmembrane domain 1 modified the effects of KN62, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and GW791343. Further studies, in which single amino acids were exchanged, identified amino acid 95 as being primarily responsible for the differential allosteric effects of GW791343 and, to varying degrees, the species differences in potency of SB203580 and KN62. The species selectivity of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid was affected by multiple regions of the receptor, with potency being particularly affected by the amino acid 126 but not by amino acid 95. A further region of the rat receptor (amino acids 154–183) was identified that, when inserted into the corresponding position in the human receptor, increased ATP potency 10-fold.

Conclusions:

This study has identified several key residues responsible for the species differences in antagonist effects at the P2X7 receptor and also identified a further region of the P2X7 receptor that can significantly affect agonist potency at the P2X7 receptor.  相似文献   

17.
  1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N- methylcarbamoyl)cinnamidoacetyl] -N-methylamino] benzyloxy] -2 - methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them.
  2. Both FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7×10−10 M and 1.5×10−9 M, respectively. They also displaced [3H]-BK binding to B2 receptors in human lung fibroblast IMR-90 cells, with an IC50 of 1.6×10−9 M and 9.8×10−10 M, respectively.
  3. In guinea-pig isolated ileum-preparations, FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA2 value of 9.2±0.1 (n=5) and a slope of 1.4±0.1 (n=5). On the other hand, FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD2 of 7.9±0.2 and the contraction was inhibited by a specific peptide bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) in a non-competitive manner.
  4. In IMR-90 cells, FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10−7 M) of the concentration-response curves to BK on PI hydrolysis. FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD2 of 8.4±0.1, and this effect was inhibited by Hoe 140.
  5. These results indicate that FR165649 and FR190997 are, respectively, a potent bradykinin B2 receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.
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18.

Background and purpose:

During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by α-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy.

Experimental approach:

Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. α-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists.

Key results:

α-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to α-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous α-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP.

Conclusions and implications:

Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects.  相似文献   

19.
  1. Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.
  2. Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.
  3. Ba2+ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+.
  4. Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.
  5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.
  6. Lubeluzole (3 μM), R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na+ channels in voltage-clamped chromaffin cells (INa) by around 20–30%. Ca2+ current through Ca2+ channels (ICa) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversible.
  7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.
  8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as well as the [Ca2+]i oscillations. The Ba2+ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na+/Ca2+ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.
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20.
  1. In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor.
  2. The P2Y1-receptor antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively).
  3. The selective P2YADP antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP-induced increases in [Ca2+]i.
  4. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist.
  5. The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPγS and ATP were lower in platelets.
  6. The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system.
  7. The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors.
  8. In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP-receptors.
  相似文献   

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