Cang-Bao Xu

Subarachnoid Hemorrhage Enhances Endothelin Receptor Expression and Function in Rat Cerebral Arteries.

OBJECTIVEInspired by organ culture-induced changes in the vascular endothelin (ET) receptor population, we investigated whether such changes occur in cerebral arteries in a rat subarachnoid hemorrhage (SAH) model. METHODSSAH was induced with injection of 250 &mgr;l of blood into the prechiasmatic cistern. After 2 days, the middle cerebral artery, basilar artery, and posterior communicating artery were harvested. Pharmacological studies were performed in vitro, and levels of messenger ribonucleic acid (mRNA) were quantified in real-time reverse transcriptase-polymerase chain reaction assays. RESULTSIn the middle cerebral artery and basilar artery from rats with induced SAH, enhanced biphasic responses to ET-1 were observed. The −log(50% effective concentration) value for the high-affinity phase was approximately 12, compared with approximately 8.5 for sham-operated animals. At a concentration of ET-1 of 10−9 mmol/L (approximately equal to the physiological concentration of ET-1 in the plasma), submaximal contractions of 50 to 75% of the contraction obtained through stimulation with 60 mmol/L K+ were now observed. Quantitative mRNA studies with the same arteries demonstrated significant increases in the number of copies of ETB receptor mRNA but not ETA receptor mRNA. Evidence of functional ETB receptors was provided in antagonist studies. The posterior communicating artery did not exhibit significant changes. CONCLUSIONThe altered receptor profile observed may represent the final stage in the series of events leading from SAH to actual spasm of the artery. The pharmacological data for the ETB receptor suggest complex interactions between normally present ETA receptors and up-regulated ETB receptors.

ERK1/2 inhibition attenuates cerebral blood flow reduction and abolishes ET(B) and 5-HT(1B) receptor upregulation after subarachnoid hemorrhage in rat.

Upregulation of endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT1B) receptors via transcription has been found after experimental subarachnoid hemorrhage (SAH), and this is associated with enhanced phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1/2). In the present study, we hypothesized that inhibition of ERK1/2 alters the ETB and 5-HT1B receptor upregulation and at the same time prevents the sustained cerebral blood flow (CBF) reduction associated with SAH. The ERK1/2 inhibitor SB386023-b was injected intracisternally in conjunction with and after the induced SAH in rats. At 2 days after the SAH, cerebral arteries were harvested for quantitative real-time polymerase chain reaction, immunohistochemistry and analysis of contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) in a sensitive myograph. To investigate if ERK1/2 inhibition had an influence on the local and global CBF after SAH, an autoradiographic technique was used. At 48 h after induced SAH, global and regional CBF were reduced by 50%. This reduction was prevented by treatment with SB386023-b. The ERK1/2 inhibition also decreased the maximum contraction elicited by application of ET-1 and 5-CT in cerebral arteries compared with SAH. In parallel, ERK1/2 inhibition downregulated ETB and 5-HT1B receptor messenger ribonucleic acid and protein levels compared with the SAH. Cerebral ischemia after SAH involves vasoconstriction and subsequent reduction in the CBF. The results suggest that ERK1/2 inhibition might be a potential treatment for the prevention of cerebral vasospasm and ischemia associated with SAH.

Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries

This study examines the importance of mitogen‐activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin‐1 (ET‐1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real‐time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c‐induced contraction. The ET‐1‐induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.

Protection against myocardial ischaemia/reperfusion injury by PPAR–α activation is related to production of nitric oxide and endothelin–1

BackgroundLigands of peroxisome proliferator–activated receptor alpha (PPAR–α) have been shown to reduce ischaemia/reperfusion injury. The mechanisms behind this effect are not well known. We hypothesized that activation of PPAR–α exerts cardioprotection via a mechanism related to nitric oxide (NO) and endothelin–1 (ET–1).MethodsFive groups of anaesthetized open–chest Sprague–Dawley rats were given the PPAR–α agonist WY 14643 1 mg/kg (WY; n = 7), dimethyl sulfoxide (DMSO, vehicle for WY; n = 6), the combination of WY and the NO synthase inhibitor N–nitro–L–arginine (L–NNA, 2 mg/kg) (n = 7), L–NNA only (n = 8) or 0.9% sodium chloride (NaCl, vehicle for DMSO and L–NNA; n = 8) i.v. before a 30 min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), eNOS and iNOS protein and ET–1 mRNA expression were determined.ResultsThere were no haemodynamic differences between the groups during the experiment. The IS was 78 ± 3% of the area at risk in the DMSO group and 77 ± 2% in the NaCl group (P = NS). WY reduced IS to 56 ± 3% (P < 0.001 vs. DMSO group). When WY was administered in combination with L–NNA the cardioprotective effect was abolished (IS 73 ± 3%, P < 0.01 vs. WY 14643). L–NNA did not affect IS per se (78 ± 2%, P = NS). The expression of eNOS but not iNOS protein in ischaemic myocardium from rats was increased in the group given WY (P < 0.05). ET–1 mRNA levels were lower in the ischaemic myocardium following WY administration.ConclusionThe results suggest that the PPAR–α activation protects the rat myocardium against ischaemia/ reperfusion injury via a mechanism related to production of NO, and possibly ET–1.

Lipid-Soluble Smoke Particles Upregulate Vascular Smooth Muscle ETB Receptors via Activation of Mitogen-Activating Protein Kinases and NF-kappaB Pathways

Cigarette smoke is a strong risk factor for cardiovascular disease. However, the underlying molecular mechanisms that lead to cigarette smoke-associated cardiovascular disease remain elusive. With functional and molecular methods, we demonstrate for the first time that lipid-soluble cigarette smoke particles (dimethylsulfoxide-soluble cigarette smoke particles; DSP) increased the expression of endothelin type B (ET(B)) receptors in arterial smooth muscle cells. The increased ET(B) receptors in arterial smooth muscle cells was documented as enhanced contractility (sensitive myograph technique), elevated levels of ET(B) receptor mRNA (quantitative real-time PCR), and protein expressions (immunohistochemistry and Western blotting). Intracellular signaling was studied with Western blotting and phosphoELISA; this revealed that DSP induced extracellular-regulated protein kinases 1 and 2 (ERK1/2), p38, and nuclear factor-kappaB (NF-kappaB) phosphorylation within 3 h. Blocking ERK1/2, p38, or NF-kappaB activation by their specific inhibitors significantly attenuated the DSP-induced upregulation of ET(B) receptor-mediated contraction and both ET(B) receptor mRNA and protein expression. In addition, dexamethasone abolished the DSP-induced upregulation of ET(B) receptor-mediated contraction. In conclusion, upregulation of ET(B) receptors by DSP in arterial smooth muscle cells involves activation of mitogen-activating protein kinases (ERK1/2 and p38) and the downstream transcriptional factor NF-kappaB pathways.

Lipid-soluble smoke particles damage endothelial cells and reduce endothelium-dependent dilatation in rat and man

BackgroundCigarette smoking is a strong risk factor for vascular disease and known to cause dysfunction of the endothelium. However, the molecular mechanisms involved are still not fully understood.MethodsIn order to reveal the direct effects of lipid-soluble smoke particles on the endothelium, ring segments isolated from rat mesenteric arteries and human middle cerebral arteries (MCA) obtained at autopsy were incubated for 6 to 48 hrs in the presence of dimethylsulphoxide (DMSO)-soluble particles from cigarette smoke (DSP), i.e. lipid-soluble smoke particles. The endothelial microstructure was examined by transmission electron microscopy. The endothelial function was evaluated by acetylcholine (ACh)-induced endothelium-dependent vasodilatation, using a sensitive myograph.ResultsAfter DSP treatment, the arterial endothelium was swollen and loosing its attachment. In functional tests, the total ACh-induced dilatation, the nitric oxide (NO)-mediated and the endothelium-derived hyperpolarization factor (EDHF)-mediated dilatations were significantly decreased by DSP in a time- and concentration-dependent manner (p < 0.05). Nicotine, an important compound in cigarette smoke had, in an equivalent concentration as in DSP, no such effects (p > 0.05). Similar results were obtained in the human MCA.ConclusionThus, we demonstrate that the lipid-soluble smoke particles, but not nicotine, caused damage to arterial endothelium and reduced the endothelium-dependent dilatation in man and rat.

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

Organ culture has been shown to upregulate both endothelin (ET) and 5‐hydroxytryptamine 1B/1D (5‐HT1B/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. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31‐7549 (specific inhibitor of classical PKCs) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET‐1 (unspecific ETA and ETB agonist), S6c (specific ETB agonist) and 5‐CT (5‐HT1 agonist). Levels of mRNA coding for the ETA, ETB, 5‐HT1B and 5‐HT1D receptors were analysed using real‐time RT–PCR. Classical PKCs are critically involved in the appearance of the ETB receptor; co‐culture with RO 31‐7549 abolished the contractile response (6.9±1.8%) and reduced the ETB receptor mRNA by 44±4% as compared to the cultured control. Correlation between decreased ETB receptor mRNA and abolished contractile function indicates upstream involvement of PKC. Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7–25% increase in Emax in response to ET‐1, S6c and 5‐CT as compared to the cultured control. Staurosporine inhibited the culture induced upregulation of the response of both the ETA and the 5‐HT1B/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.

Cigarette smoke and platelet-activating factor receptor dependent adhesion of Streptococcus pneumoniae to lower airway cells

Background Exposure to cigarette smoke (CS) is associated with increased risk of pneumococcal infection. The mechanism for this association is unknown. We recently reported that the particulate matter from urban air simulates platelet-activating factor receptor (PAFR)-dependent adhesion of pneumococci to airway cells. We therefore sought to determine whether CS stimulates pneumococcal adhesion to airway cells. Methods Human alveolar (A549), bronchial (BEAS2-B), and primary bronchial epithelial cells (HBEpC) were exposed to CS extract (CSE), and adhesion of Streptococcus pneumoniae determined. The role of PAFR in mediating adhesion was determined using a blocker (CV-3988). PAFR transcript level was assessed by quantitative real-time PCR, and PAFR expression by flow cytometry. Lung PAFR transcript level was assessed in mice exposed to CS, and bronchial epithelial PAFR expression assessed in active-smokers by immunostaining. Results In A549 cells, CSE 1% increased pneumococcal adhesion (p<0.05 vs control), PAFR transcript level (p<0.01), and PAFR expression (p<0.01). Pneumococcal adhesion to A549 cells was attenuated by CV-3988 (p<0.001). CSE 1% stimulated pneumococcal adhesion to BEAS2-B cells and HBEpC (p<0.01 vs control). CSE 1% increased PAFR expression in BEAS2-B (p<0.01), and in HBEpC (p<0.05). Lung PAFR transcript level was increased in mice exposed to CS in vivo (p<0.05 vs room air). Active smokers (n=16) had an increased percentage of bronchial epithelium with PAFR-positive cells (p<0.05 vs never smokers, n=11). Conclusion CSE stimulates PAFR-dependent pneumococcal adhesion to lower airway epithelial cells. We found evidence that CS increases bronchial PAFR in vivo.

Cigarette smoking leads to reduced relaxant responses of the cutaneous microcirculation

Background Smoking is a major risk factor for cardiovascular disease. The present study was undertaken to examine if cigarette smoking translates into reduced relaxant responses of the peripheral microcirculation. Methods The cutaneous forearm blood flow was measured by laser Doppler flowmetry. The vasodilator response to the iontophorectic administration of acetylcholine (ACh), acting via an endothelial mechanism, and sodium nitroprusside (SNP), and acting via a smooth muscle mechanism were studied. The study population consisted of 17 nonsmokers and 17 current smokers (mean age 64 ± 2 years, 13 females and 4 males) in each matched group. Results There was no difference between the groups in baseline characteristics or in basal flow. Smokers showed however significantly reduced responses to both ACh (mean ± SEM, from 973 ± 137% in nonsmokers to 651 ± 114% in smokers, p < 0.05) and SNP (from 575 ± 111% in nonsmokers to 355 ± 83% in smokers, p < 0.05). The response to the local heating (44 °C) was reduced in smokers (from 1188 ± 215% in nonsmokers to 714 ± 107% in smokers, p < 0.01). In addition, there was no difference between men and women within the groups. Conclusions The data show that cigarette smoking results in reduced peripheral microvascular responses to both endothelial and smooth muscle cell stimulation in healthy subjects, suggesting a generalized microvascular vasomotor function.

Protein kinase C inhibition prevents upregulation of vascular ET(B) and 5-HT(1B) receptors and reverses cerebral blood flow reduction after subarachnoid haemorrhage in rats.

The pathogenesis of cerebral ischaemia after subarachnoid haemorrhage (SAH) still remains elusive. The purpose of the present study was to examine whether specific protein kinas C (PKC) inhibition in rats could alter the transcriptional SAH induced Endothelin (ET) type B and 5-hydroxytryptamine type 1B (5-HT1B) receptor upregulation and prevent the associated cerebral blood flow (CBF) reduction. The PKC inhibitor RO-31-7549 or vehicle was injected intracisternally after the induced SAH in rats (n = 3 to 10 in each groups for each method). The involvement of the PKC isoforms was investigated with Western blot; only PKCδ and PKCα subtypes were increased after SAH RO-31-7549 treatment abolished this. At 2 days after the SAH basilar and middle cerebral arteries were harvested and the contractile response to endothelin-1 (ET-1; ETA and ETB receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) were investigated with a myograph. The contractile responses to ET-1 and 5-CT were increased (P < 0.05) after SAH compared with sham operated rats. In parallel, the ETB and 5-HT1B receptor mRNA and protein expression were significantly elevated after SAH, as analysed by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Administration of RO-31-7549 prevented the upregulated contraction elicited by application of ET-1 and 5-CT in cerebral arteries and kept the ETB and 5-HT1B receptor mRNA and protein levels at pre-SAH levels. Regional and global CBF evaluated by an autoradiographic technique were reduced by 60%±4% after SAH (P < 0.05) and prevented by treatment with rO-31-7549. Our study suggests that PKC plays an important role in the pathogenesis of cerebral ischaemia after SAH.