Cigarette smoke upregulates rat coronary artery endothelin receptors in vivo

Background

Cigarette smoking is a strong cardiovascular risk factor and endothelin (ET) receptors are related to coronary artery diseases. The present study established an in vivo secondhand smoke (SHS) exposure model and investigated the hypothesis that cigarette smoke induces ET receptor upregulation in rat coronary arteries and its possible underlying mechanisms.

Methodology/Principal Findings

Rats were exposed to SHS for 200 min daily for 8 weeks. The coronary arteries were isolated and examined. The vasoconstriction was studied by a sensitive myograph. The expression of mRNA and protein for receptors was examined by real-time PCR, Western blot and immunofluorescence. Compared to fresh air exposure, SHS increased contractile responses mediated by endothelin type A (ET_A) and type B (ET_B) receptors in coronary arteries. In parallel, the expression of mRNA and protein for ET_A and ET_B receptors of smoke exposed rats were higher than that of animals exposed to fresh air, suggesting that SHS upregulates ET_A and ET_B receptors in coronary arteries in vivo. Immunofluorescence staining showed that the enhanced receptor expression was localized to the smooth muscle cells of coronary arteries. The protein levels of phosphorylated (p)-Raf-1 and p-ERK1/2 in smoke exposed rats were significantly higher than in control rats, demonstrating that SHS induces the activation of the Raf/ERK/MAPK pathway. Treatment with Raf-1 inhibitor GW5074 suppressed SHS-induced enhanced contraction mediated by ET_A receptors, and inhibited the elevated mRNA and protein levels of ET_A and ET_B receptors caused by SHS. The results of correlation and regression analysis showed that phosphorylation of Raf and ERK1/2 were independent determinants to affect protein expression of ET_B and ET_A receptors.

Conclusions/Significance

Cigarette smoke upregulates ET_B and ET_A receptors in rat coronary artery, which is associated with the activation of the Raf/ERK/MAPK pathway.     

Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

Background  

Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_A and ET_B receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.     

Male-Female Differences in Upregulation of Vasoconstrictor Responses in Human Cerebral Arteries

Background and purpose

Male-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation.

Experimental approach

Freshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT_1B), angiotensin II type 1 (AT₁) and endothelin-1 type A and B (ET_A and ET_B) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression.

Key results

Vascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ET_B receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT_1B, AT₁, and ET_B receptors and in local expression of Ang II after organ culture.

Conclusions and Implications

In spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.     

Cerebrospinal Fluid from Patients with Subarachnoid Haemorrhage and Vasospasm Enhances Endothelin Contraction in Rat Cerebral Arteries

Introduction

Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ET_A and ET_B receptors.

Methods

Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ET_A and ET_B receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.

Results

Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ET_A receptors and new expression of ET_B receptors was apparent; 5) reduction in the enhanced response to endothelin after ET_B blockade in the low range and after ET_A blockade in the high range of endothelin concentrations; 6) after combined ET_A and ET_B blockade a complete inhibition of endothelin contraction was observed.

Conclusions

Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ET_A and ET_B receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.     

Synthesis and evaluation of a series of 2-substituted-5-thiopropylpiperazine (piperidine)-1,3,4-oxadiazoles derivatives as atypical antipsychotics

Background

It is important to develop novel antipsychotics that can effectively treat schizophrenia with minor side-effects. The aim of our work is to develop novel antipsychotics that act on dopamine D₂ and D₃, serotonin 5-HT_1A and 5-HT_2A receptors with low affinity for the serotonin 5-HT_2C and H₁ receptors, which can effectively cure positive symptoms, negative symptoms and cognitive impairment without the weight gain side-effect.

Methodology/Principal Findings

A series of 2-substituted-5-thiopropylpiperazine (piperidine) -1,3,4-oxadiazoles derivatives have been synthesized and the target compounds were evaluated for binding affinities to D₂, 5-HT_1A and 5-HT_2A receptors. Preliminary results indicated that compounds 14, 16 and 22 exhibited high affinities to D₂, 5-HT_1A and 5-HT_2A receptors among these compounds. Further binding tests showed that compound 22 had high affinity for D₃ receptor, and low affinity for serotonin 5-HT_2C and H₁ receptors. In addition, compound 22 inhibited apomorphine-induced climbing behavior and MK-801-induced hyperactivity with no extrapyramidal symptoms liability in mice. Moreover, compound 22 exhibited acceptable pharmacokinetic properties.

Conclusions/Significance

Compound 22 showed an atypical antipsychotic activity without liability for extrapyramidal symptoms. We anticipate compound 22 to be useful for developing a novel class of drug for the treatment of schizophrenia.     

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

Background

Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.

Methodology/Principal findings

In isolated rat mesenteric resistance arteries, ET_A-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ET_A-antagonism, were observed to promote dissociation of pre-existing ET-1/ET_A-receptor complexes.

Conclusions

Irreversible binding and activation of ET_A-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.     

Endothelin-dependent vasoconstriction in human uterine artery: application to preeclampsia

Background

Reduced uteroplacental perfusion, the initiating event in preeclampsia, is associated with enhanced endothelin-1 (ET-1) production which feeds the vasoconstriction of uterine artery. Whether the treatments of preeclampsia were effective on ET-1 induced contraction and could reverse placental ischemia is the question addressed in this study. We investigated the effect of antihypertensive drugs used in preeclampsia and of ET receptor antagonists on the contractile response to ET-1 on human uterine arteries.

Methodology/Principal Findings

Experiments were performed, ex vivo, on human uterine artery samples obtained after hysterectomy. We studied variations in isometric tension of arterial rings in response to the vasoconstrictor ET-1 and evaluated the effects of various vasodilators and ET-receptor antagonists on this response. Among antihypertensive drugs, only dihydropyridines were effective in blocking and reversing the ET-1 contractile response. Their efficiency, independent of the concentration of ET-1, was only partial. Hydralazine, alpha-methyldopa and labetalol had no effect on ET-1 induced contraction which is mediated by both ET_A and ET_B receptors in uterine artery. ET receptors antagonists, BQ-123 and BQ-788, slightly reduced the amplitude of the response to ET-1. Combination of both antagonists was more efficient, but it was not possible to reverse the maximal ET-1-induced contraction with antagonists used alone or in combination.

Conclusion

Pharmacological drugs currently used in the context of preeclampsia, do not reverse ET-1 induced contraction. Only dihydropyridines, which partially relax uterine artery previously contracted with ET-1, might offer interesting perspectives to improve placental perfusion.     

Pharmacology of endothelins: vascular preparations for studying ETA and ETB receptors

Three rabbit vessels, the carotid and pulmonary arteries and the jugular vein were investigated to identify vascular monoreceptor systems (either ET_A or ET_B) to be used in structure-activity studies on endothelins and their antagonists. The RbCA has been found to behave as a monoreceptor ETA preparation, since it shows much greater sensitivity to ET-1 than to ET-3 and is insensitive to IRL 1620. The contractile response of the RbCA to ET-1 is reduced in the presence of BQ-123 but is not influenced by BQ-788. The RbPA behaves as a pure ET_B system when stimulated with the ET_B selective agonist IRL 1620. The contractile effect of IRL 1620 is reduced in the presence of BQ-788 but is not influenced by BQ-123. The RbJV responds to ET_A and to IRL 1620 with contractions that are reduced by both BQ-123 and BQ-788, respectively. The RbJV appears to be a mixed ET_A and ET_B system in which the two functional sites play an equivalent role in the stimulatory contractile response.Thus, contractile ET_A and ET_B receptors have been found in arterial and venous vessels of the rabbit and some of these vessels provide sensitive and selective (either ET_A or ET_B) preparations that appear to be adequate for pharmacological studies on ET receptor agonists or antagonists.     

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation

Endothelin type A receptor (ET_AR) plays an important role in some cardiovascular disorders where ET_AR levels are increased. However, regulatory mechanisms for ET_AR levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET_AR-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET_AR as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET_AR-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET_AR and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET_AR, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET_AR after protein synthesis inhibition as an index of a degradation rate. ET_AR was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET_AR degradation and increased the amount of Jab1 bound to ET_AR with a maximal level of 500% at 3 h. In the absence of ET-1 stimulation, the level of ET_BR was lower than that of ET_AR and the degradation rate of ET_BR was markedly faster than that of ET_AR. Notably, the amount of Jab1 bound to ET_BR and ubiquitination level of ET_BR were markedly higher than those for ET_AR. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET_AR and ET_BR by modulating ubiquitination of these receptors, leading to changes in ET_AR and ET_BR levels.     

Expressional changes in cerebrovascular receptors after experimental transient forebrain ischemia

Background

Global ischemic stroke is one of the most prominent consequences of cardiac arrest, since the diminished blood flow to the brain results in cell damage and sometimes permanently impaired neurological function. The post-arrest period is often characterised by cerebral hypoperfusion due to subacute hemodynamic disturbances, the pathophysiology of which are poorly understood. In two other types of stroke, focal ischemic stroke and subarachnoid hemorrhage, it has earlier been demonstrated that the expression of certain vasoconstrictor receptors is increased in cerebral arteries several days after the insult, a phenomenon that leads to increased contraction of cerebral arteries, reduced perfusion of the affected area and worsened ischemic damage. Based on these findings, the aim of the present study was to investigate if transient global cerebral ischemia is associated with upregulation of vasoconstrictive endothelin and 5-hydroxytryptamine receptors in cerebral arteries. Experimental transient forebrain ischemia of varying durations was induced in male wistar rats, followed by reperfusion for 48 hours. Neurological function was assessed daily by three different tests and cerebrovascular expression and contractile function of endothelin and 5-hydroxytryptamine receptors were evaluated by wire myography, immunohistochemistry and western blotting.

Results

Transient forebrain ischemia induced neurological deficits as well as functional upregulation of vasoconstrictive ET_B and 5-HT_1B receptors in cerebral arteries supplying mid- and forebrain regions. No receptor upregulation was seen in arteries supplying the hindbrain. Immunohistochemical stainings and western blotting demonstrated expressional upregulation of these receptor subtypes in the mid- and forebrain arteries and confirmed that the receptors were located in the smooth muscle layer of the cerebral arteries.

Conclusions

This study reveals a new pathophysiological aspect of global ischemic stroke, namely expressional upregulation of vasoconstrictor receptors in cerebral arteries two days after the insult, which might contribute to cerebral hypoperfusion and delayed neuronal damage after cardiac arrest.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Modulation by endothelin-1 of spontaneous activity and membrane currents of atrioventricular node myocytes from the rabbit heart

Background

The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. Although it is known that receptors for the peptide hormone endothelin-1 (ET-1) are expressed in the AVN, there is very little information available on the modulatory effects of ET-1 on AVN electrophysiology. This study characterises for the first time acute modulatory effects of ET-1 on AVN cellular electrophysiology.

Methods

Electrophysiological experiments were conducted in which recordings were made from rabbit isolated AVN cells at 35–37°C using the whole-cell patch clamp recording technique.

Results

Application of ET-1 (10 nM) to spontaneously active AVN cells led rapidly (within ∼13 s) to membrane potential hyperpolarisation and cessation of spontaneous action potentials (APs). This effect was prevented by pre-application of the ET_A receptor inhibitor BQ-123 (1 µM) and was not mimicked by the ET_B receptor agonist IRL-1620 (300 nM). In whole-cell voltage-clamp experiments, ET-1 partially inhibited L-type calcium current (I_Ca,L) and rapid delayed rectifier K⁺ current (I_Kr), whilst it transiently activated the hyperpolarisation-activated current (I_f) at voltages negative to the pacemaking range, and activated an inwardly rectifying current that was inhibited by both tertiapin-Q (300 nM) and Ba²⁺ ions (2 mM); each of these effects was sensitive to ET_A receptor inhibition. In cells exposed to tertiapin-Q, ET-1 application did not produce membrane potential hyperpolarisation or immediate cessation of spontaneous activity; instead, there was a progressive decline in AP amplitude and depolarisation of maximum diastolic potential.

Conclusions

Acutely applied ET-1 exerts a direct modulatory effect on AVN cell electrophysiology. The dominant effect of ET-1 in this study was activation of a tertiapin-Q sensitive inwardly rectifying K⁺ current via ET_A receptors, which led rapidly to cell quiescence.     

Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling

The endothelin B (ET_B) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET_B receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET_B receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET_B receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET_B receptor localised to caveolae. In caveolae-free HEK293 cells, only ET_B receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET_B receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET_B receptor. We conclude that the ET_B receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET_B receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET_B receptors are able to induce an ERK1/2 phosphorylation.     

Increased expression of vascular endothelin type B and angiotensin type 1 receptors in patients with ischemic heart disease

Background

Endothelin-1 and angiotensin II are strong vasoconstrictors. Patients with ischemic heart disease have elevated plasma levels of endothelin-1 and angiotensin II and show increased vascular tone. The aim of the present study was to examine the endothelin and angiotensin II receptor expression in subcutaneous arteries from patients with different degrees of ischemic heart disease.

Methods

Subcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ET_A) and type B (ET_B), and angiotensin type 1 (AT₁) and type 2 (AT₂) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro pharmacology.

Results

ET_A and, to a lesser extent, ET_B receptor staining was observed in the healthy vascular smooth muscle cells. The level of ET_B receptor expression was higher in patients undergoing CABG surgery (250% ± 23%; P < 0.05) and in the patients with angina pectoris (199% ± 6%; P < 0.05), than in the healthy controls (100% ± 28%). The data was confirmed by Western blotting. Arteries from CABG patients showed increased vasoconstriction upon administration of the selective ET_B receptor agonist sarafotoxin S6c, compared to healthy controls (P < 0.05). No such difference was found for the ET_A receptors. AT₁ and, to a lesser extent, AT₂ receptor immunostaining was seen in the vascular smooth muscle cells. The level of AT₁ receptor expression was higher in both the angina pectoris (128% ± 25%; P < 0.05) and in the CABG patients (203% ± 41%; P < 0.05), as compared to the healthy controls (100% ± 25%). The increased AT₁ receptor expression was confirmed by Western blotting. Myograph experiment did however not show any change in vasoconstriction to angiotensin II in CABG patients compared to healthy controls (P = n.s).

Conclusion

The results demonstrate, for the first time, upregulation of ET_B and AT₁ receptors in vascular smooth muscle cells in ischemic heart disease. These receptors may play a role in the pathophysiology of ischemic heart disease and could provide important targets for pharmaceutical interventions.     

Ligand binding study of human PEBP1/RKIP: interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry

Background

Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer''s disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown.

Methods/Findings

In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K_D for different ligands. Native mass spectrometry was used as an alternative method for measuring K_D values.

Conclusions/Significance

Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.     

A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiation Factor eIF4AI

Background

Minocycline, a second-generation tetracycline antibiotic, has potential activity for the treatment of several neurodegenerative and psychiatric disorders. However, its mechanisms of action remain to be determined.

Methodology/Principal Findings

We found that minocycline, but not tetracycline, significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration dependent manner. Furthermore, we found that the endoplasmic reticulum protein inositol 1,4,5-triphosphate (IP₃) receptors and several common signaling molecules (PLC-γ, PI3K, Akt, p38 MAPK, c-Jun N-terminal kinase (JNK), mammalian target of rapamycin (mTOR), and Ras/Raf/ERK/MAPK pathways) might be involved in the active mechanism of minocycline. Moreover, we found that a marked increase of the eukaryotic translation initiation factor eIF4AI protein by minocycline, but not tetracycline, might be involved in the active mechanism for NGF-induced neurite outgrowth.

Conclusions/Significance

These findings suggest that eIF4AI might play a role in the novel mechanism of minocycline. Therefore, agents that can increase eIF4AI protein would be novel therapeutic drugs for certain neurodegenerative and psychiatric diseases.     

Impact of ozone exposure on the response to glucocorticoid in a mouse model of asthma: involvements of p38 MAPK and MKP-1

Background

Molecular mechanisms involved in the oxidative stress induced glucocorticoids insensitivity remain elusive. The mitogen-activated protein kinase phosphatase (MKP) 1 mediates a part of glucocorticoids action and can be modified by exogenous oxidants. Whether oxidant ozone (O₃) can affect the function of MKP-1 and hence blunt the response to corticotherapy is not clear.

Methods

Here we employed a murine model of asthma established with ovalbumin (OVA) sensitization and challenge to evaluate the influence of O₃ on the inhibitory effect of dexamethasone on AHR and airway inflammation, and by administration of SB239063, a selective p38 MAPK inhibitor, to explore the underlying involvements of the activation of p38 MAPK and the expression of MKP-1.

Results

Ozone exposure not only aggravated the pulmonary inflammation and AHR, but also decreased the inhibitory effects of dexamethasone, accompanied by the elevated oxidative stress, airway neutrophilia, enhanced phosphorylation of p38 MAPK, and upregulated expression of IL-17. Administration of SB239063 caused significant inhibition of the p38 MAPK phosphorylation, alleviation of the airway neutrophilia, and decrement of the ozone-induced IL-17 expression, and partly restored the ozone-impaired effects of dexamethasone. Ozone exposure not only decreased the protein expression of MKP-1, but also diminished the dexamethasone-mediated induction process of MKP-1 mRNA and protein expression.

Conclusions

The glucocorticoids insensitivity elicited by ozone exposure on current asthma model may involve the enhanced phosphorylation of p38 MAPK and disturbed expression of MKP-1.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0126-x) contains supplementary material, which is available to authorized users.