Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

Endothelin-1 is a potent vasoconstrictor mediating its effects via two receptor subtypes, the endothelin type A (ET(A)) preferentially situated on smooth muscle cells, mediating vasoconstriction and endothelin type B (ET(B)) mainly located on endothelial cells, mediating vasodilatation. In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries but produced significant vasoconstriction after organ culture. The endothelin ET(B) receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ET(A) receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture. Pharmacological inhibition of endothelium-derived dilatory mediators did not influence endothelin ET(A) or ET(B) receptor-mediated vasoconstriction in fresh segments. In cultured arteries, inhibition of endothelial vasodilators potentiated the effect of sarafotoxin 6c. In conclusion, endothelin ET(B) receptor stimulation in cultured coronary arteries elicits vasoconstriction. This is likely not related to endothelial dysfunction with putative loss of its vasodilator components, but rather explained by the up-regulation of contractile endothelin ET(B) receptors on smooth muscle cells.     

Protein kinase mediated upregulation of endothelin A,endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery

1. Organ culture has been shown to upregulate both endothelin (ET) and 5-hydroxytryptamine 1B/1D (5-HT(1B/1D)) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process. 2. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhibitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET-1 (unspecific ET(A) and ET(B) agonist), S6c (specific ET(B) agonist) and 5-CT (5-HT(1) agonist). Levels of mRNA coding for the ET(A), ET(B), 5-HT(1B) and 5-HT(1D) receptors were analysed using real-time RT-PCR. 3. Classical PKC's are critically involved in the appearance of the ET(B) receptor; co-culture with RO 31-7549 abolished the contractile response (6.9 +/- 1.8%) and reduced the ET(B) receptor mRNA by 44 +/- 4% as compared to the cultured control. Correlation between decreased ET(B) receptor mRNA and abolished contractile function indicates upstream involvement of PKC. 4. Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7-25% increase in E(max) in response to ET-1, S6c and 5-CT as compared to the cultured control. 5. Staurosporine inhibited the culture induced upregulation of the response of both the ET(A) and the 5-HT(1B/1D) receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases.     

Amphetamine increases phosphorylation of extracellular signal-regulated kinase and transcription factors in the rat striatum via group I metabotropic glutamate receptors.

Amphetamine is an indirect dopamine receptor agonist and increases glutamate release in the striatum. Activation of group I metabotropic glutamate receptors (mGluRs) upregulates cAMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1 and 2 (ERK1/2) in the striatum in vivo. In the present study the role of mGluRs in the regulation of ERK1/2 pathways leading to CREB and Elk-1 phosphorylation by amphetamine was investigated using immunohistochemistry and Western blot in the rat dorsal striatum. Acute administration of amphetamine (5 mg/kg, i.p.) caused increases in phosphorylated (p)CREB, pElk-1, and pERK1/2 immunoreactivity. Intrastriatal blockade of group I mGluRs with N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) significantly attenuated amphetamine-induced pCREB, pElk-1, pERK1/2, and Fos immunoreactivity in both medial and lateral areas of the striatum. Systemic injection of an mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 10 mg/kg, i.p.), also blocked the amphetamine induction of these phosphoproteins. In contrast, intrastriatal blockade of group II/III mGluRs with (RS)-alpha-methylserine-o-phosphate monophenyl ester (MSOPPE; 25 nmol) did not affect amphetamine-induced increases in all the four markers. Similarly, intrastriatal dantrolene (2 or 20 nmol) that blocks intracellular Ca(2+) release from ryanodine-sensitive stores did not affect amphetamine effects. Injection of PHCCC, MPEP, MSOPPE, or dantrolene alone did not alter basal levels of the three phosphoproteins and Fos. These data suggest that acute amphetamine is able to facilitate the phosphorylation of CREB, Elk-1, and ERK1/2 signaling proteins and Fos gene expression via a group I mGluR-dependent mechanism in the dorsal striatum.     

辛伐他汀改善大鼠心肌梗死后心室重塑与p-ERK1/2的关系

目的探讨辛伐他汀对大鼠心肌梗死后心室重塑的影响及其与磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的关系。方法结扎Wistar大鼠冠状动脉左前降支建立心肌梗死模型,大鼠随机分为4组(n=8~10):心肌梗死对照组、辛伐他汀20、40mg处理组和假手术组。4wk后心脏超声检测各组心脏形态和功能,免疫组化法和Western blot检测p-ERK1/2表达。结果与假手术组相比,心肌梗死对照组及辛伐他汀处理组左室舒张末期内径(LVEDd)、左室后壁厚度(LVPWd)明显增加(P<0.01),左室射血分数(LVEF)、短轴缩短率(FS)、每博输出量(SV)和心输出量(CO)明显降低(P<0.01),心肌p-ERK1/2表达明显升高(P<0.01)。与心肌梗死对照组相比,辛伐他汀处理组LVEDd、LVPWd明显减少(P<0.01),LVEF、FS、SV和CO明显升高(P<0.01),心肌p-ERK1/2表达明显下降(P<0.01);其中辛伐他汀40mg组比20mg组心肌p-ERK1/2表达下降更明显(P<0.05)。结论辛伐他汀改善心肌梗死后心室重塑和心功能,其机制可能与下调心肌p-ERK1/2表达有关。     

Atorvastatin attenuates vascular remodelling in spontaneously hypertensive rats via the protein kinase D/extracellular signal-regulated kinase 5 pathway

The present study was conducted to determine whether atorvastatin reduces hypertension-induced vascular remodelling and whether its effects involve protein kinase D (PKD) and extracellular signal-regulated kinase 5 (ERK5). We used 16-week-old spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. The blood pressure and serum lipid concentration were measured. Changes in the vascular morphology and histology were examined using H&E, Masson^’s trichrome, and Sirius Red staining. The media thickness (MT), ratio of MT to lumen diameter (LD) (MT/LD), collagen volume fraction (CVF) and hydroxyproline content were measured to evaluate vascular remodelling. Atorvastatin (50 mg/kg/day) was administered for 8 weeks. Increased blood pressure and vascular remodelling were more prominent in SHRs than in WKY rats. SHRs also had elevated PKD and ERK5 activation. The systolic blood pressure, MT/LD ratio, and hydroxyproline content were positively correlated with the activation level of PKD and ERK5 in SHRs. Atorvastatin significantly attenuated the activation of PKD and ERK5. Overall, this study demonstrated that atorvastatin could reverse vascular remodelling in SHRs. The PKD/ERK5 signalling pathway might be important for elucidating the beneficial pleiotropic effects of atorvastatin on vascular remodelling.     

Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists

Agonist activity at recombinant human dopamine D_4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [³⁵S]GTPγS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [³⁵S]GTPγS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [³⁵S]GTPγS), selected compounds were tested in a modified short-duration [³⁵S]GTPγS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D₄ receptors in vivo. This paper is especially dedicated to the memory of our appreciated colleague and co-author, Dr. Liesbeth Bruins Slot, who left us much too soon the 4th of April, 2008.     

Role of mitogen-activated protein kinases in endothelin ETB receptor up-regulation after organ culture of rat mesenteric artery

Organ culture of isolated arteries results in increased levels of endothelin ET(B) (ET(B)) receptor mRNA and in enhanced ET(B) receptor mediated contraction. The present study was designed to pinpoint the mitogen-activated protein kinase (MAPK) subtype involved in up-regulation of ET(B) receptors after organ culture of rat mesenteric arteries. Western blot and selective antibodies towards constitutional and phosphorylated MAPKs revealed the appearance of phosphorylated MAPK of the extracellular signal-regulated kinases (ERK) 1/2 type at 3 h of organ culture. The functional ET(B) receptor and its mRNA expression were up-regulated after 24 h of organ culture. Following incubation with the MEK 1/2 specific inhibitor SB408039 or the raf inhibitor SB386023b the up-regulation was attenuated both for ET(B) receptor responses and in ET(B) receptor mRNA expression in the vessel segments. Neither Western blot nor myograph or mRNA analysis showed involvement of the other MAPKs studied. Our results suggest that the ERK1/2 MAPKs are involved in the endothelin ET(B) receptor up-regulation following organ culture.     

Influence of AMPA/kainate receptors on extracellular 5-hydroxytryptamine in rat midbrain raphe and forebrain

1. The regulation of 5-hydroxytryptamine (5-HT) release by excitatory amino acid (EAA) receptors was examined by use of microdialysis in the CNS of freely behaving rats. Extracellular 5-HT was measured in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), nucleus accumbens, hypothalamus, frontal cortex, dorsal and ventral hippocampus.
2. Local infusion of kainate produced increases in extracellular 5-HT in the DRN and MRN. Kainate infusion into forebrain sites had a less potent effect.
3. In further studies of the DRN and nucleus accumbens, kainate-induced increases in extracellular 5-HT were blocked by the EAA receptor antagonists, kynurenate and 6,7-dinitroquinoxaline-2,3-dione (DNQX).
4. The effect of infusing kainate into the DRN or nucleus accumbens was attenuated or abolished by tetrodotoxin (TTX), suggesting that the increase in extracellular 5-HT is dependent on 5-HT neuronal activity. In contrast, ibotenate-induced lesion of intrinsic neurones did not attenuate the effect of infusing kainate into the nucleus accumbens. Thus, the effect of kainate in the nucleus accumbens does not depend on intrinsic neurones.
5. Infusion of α-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate (AMPA) into the DRN and nucleus accumbens induced nonsignificant changes in extracellular 5-HT. Cyclothiazide and diazoxide, which attenuate receptor desensitization, greatly enhanced the effect of AMPA on 5-HT in the DRN, but not in the nucleus accumbens.
6. In conclusion, AMPA/kainate receptors regulate 5-HT in the raphe and in forebrain sites.

     

Different effect of ouabain on endothelin-1-induced extracellular signal-regulated kinase stimulation in rat heart and tail artery

Endogenous ouabain may play a role in the control of cardiovascular system function. In this study, we investigated the effects of a long-term ouabain treatment on basal and endothelin-1 (ET-1)-induced phosphorylation of cardiac and vascular extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), which are involved in several cardiac and vascular physiologic and pathologic conditions. Our results show that the hearts from ouabain-treated rats have a higher basal level of ERK-1 and ERK-2 phosphorylation compared with untreated rats. Perfusion of the hearts with ET-1 increased ERK-1 and ERK-2 phosphorylation both in ouabain-treated and in control rats, with a larger stimulatory effect in ouabain-treated animals. On the contrary, exposure of endothelium-free tail artery to ET-1 increased ERKs phosphorylation both in treated and untreated rats, but this effect was blunted in ouabain-treated rats. These findings demonstrate that ouabain treatment has opposite effects on basal and ET-1-induced ERKs phosphorylation in the heart and in the tail artery of the rat.     

5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

5-Hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells are extracellular signal-regulated kinase pathway dependent

AIM: To investigate the effect of 5-hydroxytryptamine transporter (5-HTT) inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN) to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. METHODS: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate (FITC)-labeled antisense ODN in PASMCs. The effects of 5-HTT selective inhibitor fluoxetine and ODNs on the proliferation of PASMCs were evaluated by cell number counting and cell cycle analysis, and measured by microculture tetrazolium (MTT) assay and flow cytometry (FCM), respectively. RESULTS: Liposomes mediated the transfection of ODNs into PASMCs with high efficiency. MTT assay showed fluoxetine (10 micromol/L, 1 micromol/L, and 100 nmol/L) concentration dependently inhibited the proliferation of PASMCs induced by 5-HT (1 micromol/L) in vitro. The proliferation rate of PASMCs by 5-HT was significantly inhibited by pretreatment with ERK1/2 antisense ODN (0.2 micromol/L) from 251%+/-18% to 86%+/-5% (P<0.01). Flow cytometric analysis of cell cycle distribution showed that the increase of 5-HT induced S phase fraction (SPF) and proliferation index (PI) were significantly inhibited by fluoxetine (1 micromol/L) or antisense ODN with SPF from 36%+/-4% to 26%+/-3% and 24%+/-4%, and PI from 34%+/-2% to 29%+/-2% and 24%+/-2%, respectively. CONCLUSION: 5-HTT mediates the mitogenic effect of 5-HT on PASMCs and the proliferation of PASMCs induced by 5-HT is dependent on ERKs signal pathway.     

Involvement of YC-1 in extracellular signal-regulated kinase action in rat cremasteric muscle

Objectives The nitric oxide (NO)–soluble guanylate cyclase (sGC) signalling pathway is attributed to the prevention of ischaemia–reperfusion (I/R)‐induced leucocyte–endothelium adhesive interactions. YC‐1 (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole), a NO‐independent sGC activator, has been shown to exert cardiovascular benefits, but its action on leucocyte–endothelium interactions remains unknown. In this study, the direct effect and the underlying mechanism of the anti‐adhesive action of YC‐1 have been examined in cremasteric microcirculation. Methods Rat cremaster muscle was subjected to 4 h pudic‐epigastric artery ischaemia followed by 2 h reperfusion and intravital microscopy was used to observe leucocyte–endothelium interaction and to quantify functional capillaries in rat cremaster muscle flaps. Key findings The values for leucocyte rolling, adhering and transmigrating were 5.5‐, 6.9‐ and 8.8‐fold greater, respectively, in I/R than in sham‐control animals. YC‐1 treatment rescued functional capillary density and reduced leucocyte rolling, adhering and transmigrating in I/R injured cremaster muscles to levels observed in sham‐controls. Interestingly, these effects were completely blocked by the MEK (extracellular signal‐regulated kinase (ERK) kinase) inhibitor (PD98059) but not by sGC or protein kinase C inhibitors. Cotreatment of PD98059 with YC‐1 caused a 3.3‐, 7.5‐ and 8.3‐fold increase in the values for leucocyte rolling, adhering and transmigrating, respectively, in postcapillary venules of I/R‐injured cremaster muscle. Conclusions This study has indicated that the anti‐adhesive and functional capillary density rescue properties of YC‐1 were mediated predominantly by the activation of ERK but not sGC, although YC‐1 was identified to be a sGC activator. A better understanding of the action of YC‐1 on the microvasculature may help shed light on its therapeutic potential for cardiovascular disease.     

5-HT1B receptors mediate potent contractile responses to 5-HT in rat caudal artery.

5-Hydroxytryptamine (5-HT) evoked potent contractile responses in phenoxybenzamine-treated ring segments of rat caudal artery, partially contracted with U46619. Responses were mimicked by 5-HT1-selective agonists with the potency order: RU24969 > 5-carboxamidotryptamine > 5-HT = CP-93,129 >> sumatriptan. 8-Hydroxy-N,N-dipropylaminotetralin was virtually inactive. Responses were unaffected by spiperone (0.1 microM) and mesulergine (1.0 microM), but were antagonized competitively by (+/-)-cyanopindolol affording agonist-independent pKB estimates of 8.4 to 8.9. The pharmacological profile of this receptor is consistent with that of the 5-HT1B subtype. Since the 5-HT1B receptor is the rodent homologue of the 5-HT1D beta subtype, it might be anticipated that 5-HT1D beta receptors will be found to mediate vasoconstrictor responses in non-rodent species.     

Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in rat soleus muscle

1. The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the extracellular-signal regulated protein kinase (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) pathways was determined in rat muscle. 2. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary (NT; n = 8); (ii) low-intensity training (8 m/min; LIT; n = 16); and (iii) moderate-to-high-intensity training (28 m/min; HIT; n = 16). The training regimens were planned so that animals covered the same distance and had similar glycogen utilization for both LIT and HIT exercise sessions. 3. A single bout of LIT or HIT following 8 weeks of training led to a twofold increase in the phosphorylation of ERK1/2 (P = 0.048) and a two- to threefold increase in p38 MAPK (P = 0.005). Extracellular signal-regulated kinase 1/2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAPK phosphorylation was 70-80% lower than sedentary. One bout of LIT or HIT increased total ERK1/2 and p38 MAPK expression, with the magnitude of this increase being independent of prior exercise intensity or duration. Extracellular signal- regulated kinase 1/2 expression was increased three- to fourfold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P = 0.027), but p38 MAPK expression was approximately 90% lower than sedentary values. 4. In conclusion, exercise-training of different intensities/ durations results in selective postexercise activation of intracellular signalling pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes.     

Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110gamma/Raf-1 and protein kinase A/B-Raf signaling in human skin fibroblasts

Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappaE), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.