Mikael Adner

Cholesterol Depletion Impairs Vascular Reactivity to Endothelin-1 by Reducing Store-Operated Ca2+ Entry Dependent on TRPC1

Abstract— The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-&bgr;-cyclodextrin (m&bgr;cd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by m&bgr;cd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by m&bgr;cd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in m&bgr;cd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase–polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by m&bgr;cd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1–induced contractions were insensitive to the TRPC1 blocking antibody and to m&bgr;cd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to m&bgr;cd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.

Plasticity of contractile endothelin‐B receptors in human arteries after organ culture

1 The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase‐polymerase chain reaction (RT‐PCR). 2 In freshly obtained segments of human omental arteries, ET‐1 and ET‐3 induced concentration‐dependent contractions which were normalized to the response produced by 60 mM K+. ET‐1 produced a maximum contraction (Emax) amounting to 151 ± 17% of the K+ response. The pEC50 for this agonist was 8.64 ± 0.17. The effect of ET‐3 was less pronounced (Emax: 71 ± 22% and pEC50: 6.69 ± 0.17) than that of ET‐1. The ET receptors involved were characterized with FR139317 (a selective ETA receptor antagonist), PD 145065 (a mixed ETA and ETB receptor antagonist) and BQ 788 (an ETB receptor antagonist). A high concentration of these antagonists (10 μm) abolished the contractile responses to ET‐3, and produced a parallel rightward shift of the ET‐1 concentration‐response curve without changing the maximal effect. FR 139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ETA receptors mediating contraction in human omental arteries. 3 Arterial segments cultured for 5 days in serum‐free Dulbeccos medium at 37°C under sterile and humidified conditions retained contractility although responses to 60 mM K+ were somewhat reduced. ET‐3 was significantly more potent in the cultured arteries (pEC50: 8.56 ± 0.15) and achieved a greater maximum effect (Emax: 116 ± 19%). Responses were not antagonised by FR 139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast Emax (179 ± 17%) and pEC50 (8.66 ± 0.23) values for ET‐1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET‐1‐induced concentration‐response curve, whereas FR 139317 and BQ 788 caused non‐significant shifts. These findings suggest that functional ETB receptors contribute significantly to the endothelin contractile response in cultured arteries. 4 Two‐site analysis of the ET‐1 induced concentration‐response curve from cultured arteries suggests that ETB receptors, at the high potency component, and ETA receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ETA receptor would be capable of evoking at least 75% of the ET‐1 contraction in the absence of ETB receptors, although with a lower potency as compared to fresh arteries. 5 Electrophoresis of RT‐PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ETA and ETB receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ETB receptor as compared to the ETA receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6 In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ETA receptor subtype, with a negligible contribution by ETB receptors. Cultured arterial segments, however, exhibited a substantial ETB receptor mediated contractile response and an increase in ETB receptor mRNA content, consistent with an upregulation of functional ETBreceptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ETBreceptors.

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by Pam3CSK4, R-837 and CpG-2006 stimulation

Toll‐like receptors (TLRs) recognize specific pathogen‐associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1–TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19+ CD38– CD27– (naïve B cells), CD19+ IgD– CD27–[germinal centre (GC) B cells] and CD19+ CD38– CD27+ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo‐isolated B‐cell subsets. Purified CD19+ B cells stimulated with Pam3CSK4, R‐837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin‐6 secretion and an up‐regulated expression of human leucocyte antigen (HLA)‐DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B‐cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.

Toll-like receptor stimulation induces airway hyper-responsiveness to bradykinin, an effect mediated by JNK and NF-κB signaling pathways

Airway infections induce hyper‐responsiveness in asthmatic patients. Toll‐like receptors (TLR) mediate inflammatory responses to microbes. Occurrence and effects of TLR2, TLR3 and TLR4 were examined in a mouse organ culture model of asthma focusing on the smooth muscle responses to bradykinin. TLR2, TLR3 and TLR4 mRNA, and TLR2 and TLR4 immunoreactivity were detected in the tracheal muscle layer. Tracheal organ culture for 1 or 4 days with lipopolysaccharide (LPS; TLR2/4 agonist) or polyinosinic polycytidylic acid (poly‐I‐C; TLR3 agonist) enhanced bradykinin‐ and [des‐Arg9]‐bradykinin‐induced contractions. Simultaneous LPS and poly‐I‐C treatment resulted in synergistic enhancement of bradykinin‐induced contraction. In carbachol‐pre‐contracted segments TLR stimulationinduced less potent relaxations to bradykinin and [des‐Arg9]‐bradykinin. The LPS and poly‐I‐C enhancement of bradykinin‐induced contraction was inhibited by the transcriptional inhibitor actinomycin‐D, dexamethasone, the proteasome inhibitor MG‐132 and the c‐Jun N‐terminal kinase (JNK) inhibitor SP600125. LPS and poly‐I‐C induced translocation of NF‐κB p65 to the nucleus and up‐regulation of kinin B1 and B2 receptor mRNA. In summary, TLR2, TLR3 and TLR4 are expressed in the mouse tracheal smooth muscle. Costimulation of these receptors results in NF‐κB‐ and JNK‐mediated transcription of B1 and B2 receptor, inducing hyper‐responsiveness to bradykinin.

Up-regulation of Toll-like receptors 2, 3 and 4 in allergic rhinitis

BackgroundToll-like receptors enable the host to recognize a large number of pathogen-associated molecular patterns such as bacterial lipopolysaccharide, viral RNA, CpG-containing DNA and flagellin. Toll-like receptors have also been shown to play a pivotal role in both innate and adaptive immune responses. The role of Toll-like receptors as a primary part of our microbe defense system has been shown in several studies, but their possible function as mediators in allergy and asthma remains to be established. The present study was designed to examine the expression of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with intermittent allergic rhinitis, focusing on changes induced by exposure to pollen.Methods27 healthy controls and 42 patients with seasonal allergic rhinitis volunteered for the study. Nasal biopsies were obtained before and during pollen season as well as before and after allergen challenge. The seasonal material was used for mRNA quantification of Toll-like receptors 2, 3 and 4 with real-time polymerase chain reaction, whereas specimens achieved in conjunction with allergen challenge were used for immunohistochemical localization and quantification of corresponding proteins.ResultsmRNA and protein representing Toll-like receptors 2, 3 and 4 could be demonstrated in all specimens. An increase in protein expression for all three receptors could be seen following allergen challenge, whereas a significant increase of mRNA only could be obtained for Toll-like receptor 3 during pollen season.ConclusionThe up-regulation of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with symptomatic allergic rhinitis supports the idea of a role for Toll-like receptors in allergic airway inflammation.

Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis

BackgroundThe palatine tonsils have a pivotal role in immunological detection of airborne and ingested antigens like bacteria and viruses. They have recently been demonstrated to express Toll-like receptors (TLRs), known to recognize molecular structures on such microbes and activate innate immune responses. Their activation might also provide a link between innate and adaptive immunity. In the present study, the expression profile of TLR1-TLR10 was characterized in human tonsil T cells, focusing on differences between subsets of CD4+ T helper (Th) cells and CD8+ cytotoxic T lymphocytes (CTL). The study was also designed to compare the TLR expression in T cells from patients with recurrent tonsillitis and tonsillar hyperplasia.MethodsTonsils were obtained from children undergoing tonsillectomy, and classified according to the clinical diagnoses and the outcome of tonsillar core culture tests. Two groups were defined; recurrently infected tonsils and hyperplastic tonsils that served as controls. Subsets of T cells were isolated using magnetic beads. The expression of TLR transcripts in purified cells was assessed using quantitative real-time RT-PCR. The corresponding protein expression was investigated using flow cytometry and immunohistochemistry.ResultsT cells expressed a broad repertoire of TLRs, in which TLR1, TLR2, TLR5, TLR9 and TLR10 predominated. Also, a differential expression of TLRs in CD4+ and CD8+ T cells was obtained. TLR1 and TLR9 mRNA was expressed to a greater extent in CD4+ cells, whereas expression of TLR3 mRNA and protein and TLR4 protein was higher in CD8+ cells. CD8+ cells from infected tonsils expressed higher levels of TLR2, TLR3 and TLR5 compared to control. In contrast, CD4+ cells exhibited a down-regulated TLR9 as a consequence of infection.ConclusionThe present study demonstrates the presence of a broad repertoire of TLRs in T cells, a differential expression in CD4+ and CD8+ cells, along with infection-dependent alterations in TLR expression. Collectively, these results support the idea that TLRs are of importance to adaptive immune cells. It might be that TLRs have a direct role in adaptive immune reactions against infections. Thus, further functional studies of the relevance of TLR stimulation on T cells will be of importance.

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFalpha up-regulates 5-HT(2A)-mediated contraction.

Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of inflammatory mediators that affect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1–32 days, and investigated contractile responses mediated by muscarinic and 5‐hydroxytryptamine (5‐HT) receptors following long‐term exposure to tumour necrosis factor‐alpha (TNFα). Following culture, in the absence of TNFα, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not affected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 5‐HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFα produced a time‐ and concentration‐dependent increase in the maximal contraction to 5‐HT, evidently mediated by 5‐HT2A receptors, whereas, the potency for carbachol was reduced. In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the first days of culture. The time‐dependent effect of TNFα indicates the importance of studying the long‐term effect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

Presence of contractile endothelin-A and dilatory endothelin-B receptors in human cerebral arteries.

OBJECTIVE The aim of the present study was to elucidate the endothelin receptor subtypes responsible for the endothelin-induced vasomotor responses of human cerebral arteries. METHODS Human cerebral arteries with endothelium were mounted in in vitro tissue baths, and the vascular responses to endothelin-1 (ET-1) and sarafotoxin 6c (a selective ETB agonist) were studied in the presence or absence of endothelin blockers, bosentan (Ro 47-0203), a novel nonpeptide ETA and ETB receptor antagonist, and FR139317, a selective ETA receptor antagonist. The presence of messenger ribonucleic acid encoding the human ETA and ETB receptors in human cerebral arteries with intact endothelium and in segments denuded of endothelium was studied by the use of reverse transcriptase-polymerase chain reaction. RESULTS ET-1 induced concentration-dependent contraction of human cerebral arteries; the pEC50 value was 9.4 +/- 0.2. The vasoconstriction was significantly antagonized both by bosentan and by FR139317. The pA2 values were 7.2 +/- 0.4 and 7.4 +/- 0.4, respectively. Sarafotoxin 6c failed to cause contraction of human cerebral arteries. In precontracted vessels, however, sarafotoxin 6c induced dilatation that was significantly inhibited by bosentan (10 mumol/L), resulting in a pA2 value of 6.0 +/- 0.2. Furthermore, messenger ribonucleic acid encoding the human ETA and ETB receptors was detected in human cerebral arteries both with and without endothelium. CONCLUSION The ET-1-induced vasoconstriction of human cerebral arteries is primarily mediated by the ETA receptor, whereas the sarafotoxin 6c-induced vasodilatation seems to be mediated via the ETB receptor.

The stable pyrimidines UDPβS and UTPγS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery

The contractile and relaxant effects of the different P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5′‐O‐thiodiphosphate (UDPβS) and uridine 5′‐O‐3‐thiotriphosphate (UTPγS). The selective P2X receptor agonist, αβ‐methylene‐adenosine triphosphate (αβ‐MeATP) stimulated a potent (pEC50=6.0) but relatively weak contraction (Emax=57% of 60 mM K+). The contractile concentration‐response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The first part of the response could be desensitized by αβ‐MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors. The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 μM αβ‐MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1–10 mM). The selective P2Y6 agonist, UDPβS, and the P2Y2/P2Y4‐receptor agonists UTPγS and adenosine 5′‐O‐3‐thiotriphosphate (ATPγS) were considerably more potent and efficacious (Emax∼250% of 60 mM K+). Adenosine 5′‐O‐thiodiphosphate (ADPβS) was inactive, excluding contractile P2Y1 receptors. After precontraction with 1 μM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC50∼5.0). UTPγS, ADPβS and ATPγS were approximately one log unit more potent indicating the presence of endothelial P2Y1 and P2Y2/P2Y4 receptors. The P2Y6 receptor agonist, UDPβS, had no effect. UDPβS and UTPγS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y6 and, slightly less potently, P2Y2/P2Y4 receptors. The P2X response was relatively weak, and there was no P2Y1 response. Stimulation of P2Y1 and P2Y2/P2Y4 receptors elicited relaxation, while P2Y6 did not contribute.

Transcriptional regulated plasticity of vascular contractile endothelin ETB receptors after organ culture

The aim of the present study was to investigate the level of regulation of the contractile endothelin ET(B) receptor which appears spontaneously after organ culture of vascular segments. Endothelin-1 elicited a strong contraction while the selective endothelin ET(B) receptor agonist, sarafotoxin 6c, had a negligible effect on fresh ring segments of rat mesenteric artery. After organ culture in serum-free Dulbeccos modified Eagles medium at 37 degrees C (for 1 or 2 days) the endothelin-1-induced contraction was unchanged, whereas sarafotoxin 6c induced, after 1 day, a marked contraction which was further increased at day 2. The contraction induced by sarafotoxin 6c was significantly attenuated by the transcriptional inhibitor, actinomycin D, or the translational inhibitor, cyclohexamide, while the endothelin-1-induced contraction was much less affected. mRNA for endothelin ET(A) and endothelin ET(B) receptors was present in fresh human omental arteries denuded of endothelium. However, after organ culture, endothelin ET(B) mRNA was more prominent than endothelin ET(A) mRNA. Furthermore, the mRNA for both receptors was decreased after treatment with actinomycin D but not with cyclohexamide. This suggests that the endothelin ET(A) receptor is the dominating contractile receptor in fresh arteries while organ culture induces transcription and subsequent translation of contractile endothelin ET(B) receptors.