Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C > alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.     

Kupffer细胞在APAP所致肝损伤中作用研究进展

对乙酰氨基酚所致肝损伤(acetaminophen-induced liver injury, AILI)是一类普遍的药物源性肝损伤(drug-induced liver injury, DILI),是造成急性肝衰竭(acute liver failure, AIF)的主要原因。Kupffer细胞为肝脏固有巨噬细胞,是机体先天免疫重要组成部分。Kupffer具有促炎和抗炎的双重作用,通过识别损伤相关模式分子(damage-associated molecular patterns, DAMPs)激活细胞内炎症信号,释放促炎因子、抗炎因子和趋化因子。Kupffer在AILI氧化应激、细胞招募、炎症反应、肝再生和纤维化等过程起着重要作用,对AILI的发生、发展及转归有着重要的影响。     

Role of intrarenal alpha(2)-adrenoceptors in the renal responses to xylazine in rats

This study examined the contribution of intrarenal alpha(2)-adrenoceptor mechanisms to the enhanced urine flow rate (V) and urinary sodium excretion (U(Na)V) responses in ketamine-xylazine-anesthetized rats. Ten minutes after left renal artery (LRA) injection, the alpha(2)-adrenoceptor antagonist yohimbine (5 microg) significantly decreased V from 58 +/- 8 to 35 +/- 7 microl. min(-1). g kidney wt(-1) and U(Na)V from 2.8 +/- 0.4 to 2.1 +/- 0.4 microeq. min(-1). g kidney wt(-1) without altering right kidney function. The renal effects of the LRA injection of yohimbine were completely abolished in chronic bilaterally renal-denervated (RDNX) rats. In RDNX rats, a higher LRA dose of yohimbine (15 microg) significantly reduced left and right kidney V, with no effects on U(Na)V. In separate bladder-catheterized rats, yohimbine (0.5 mg/kg), 20 min after intravenous injection, significantly decreased V from 63 +/- 9 to 13 +/- 2 microl. min(-1). g kidney wt(-1 )and U(Na)V from 4.5 +/- 0.5 to 1.1 +/- 0.1 microeq. min(-1). g kidney wt(-1). In RDNX rats, this dose of yohimbine reduced V and U(Na)V, but the magnitude was blunted compared with intact rats. In contrast, 0.1 mg/kg iv yohimbine significantly reduced V and U(Na)V to similar magnitudes in intact and RDNX groups. Together, these findings indicate that intravenous xylazine acts by renal nerve-dependent and -independent mechanisms to enhance renal excretory function in ketamine-anesthetized rats. Because the effects of the LRA dose of yohimbine were abolished in renal-denervated animals, it appears that xylazine has a direct renal action to augment the renal excretion of water and sodium via a presynaptic alpha(2)-adrenoceptor pathway that inhibits the release of neurotransmitters from renal sympathetic nerve terminals.     

Adrenaline (via alpha(1B)-adrenoceptors) and ethanol stimulate OH* radical production in isolated rat hepatocytes

Adrenaline is able to increase the oxidative damage caused by some xenobiotic agents in the liver. Ethanol produces oxidative changes in hepatic tissue, while an acute intoxication with alcohol increases adrenaline blood levels. The aim of this study was to determine whether adrenaline increases ethanol-induced hydroxyl free radical production in isolated hepatocytes. Adrenaline augmented hydroxyl radicals in a concentration-dependent manner and was blocked by chloroethylclonidine, an alpha(1B)-adrenoceptor antagonist, while adrenaline plus ethanol added their individual effects. It is suggested that adrenaline increases hydroxyl radicals by an alpha(1B)-adrenoceptor-mediated mechanism, while ethanol does so by a receptor-independent mechanism.     

Leptin enhances TNF-alpha production via p38 and JNK MAPK in LPS-stimulated Kupffer cells

Leptin is now recognized as a proinflammatory cytokine and thought to be a progressive factor for non-alcoholic steatohepatitis (NASH). Here we showed the effects of leptin on the production of TNF-alpha (tumor necrosis factor-alpha) by Kupffer cells (KCs) with signal transduction. Leptin enhanced TNF-alpha production accompanied by a dose-dependent increase of MAPK activity in lipopolysaccharide (LPS)-stimulated KCs. SB203580 and JNK inhibitor I, specific inhibitors of P38 and JNK, inhibited TNF-alpha production in KCs but PD98059, an inhibitor of the ERK pathway, did not affect TNF-alpha production by KCs. Recombinant constitutively active adenovirus (Ad)-MKK6 and-MKK7 increased TNF-alpha production in KCs with activation of P38 and JNK without any change by Ad-MEK1 delivery. On the other hand, KCs isolated from the Zucker rat (fa/fa), a leptin receptor-deficient rat, showed reduced production of TNF-alpha on stimulation with LPS. The delivery of Ad-MKK6 and-MKK7, but not Ad-MEK1, increased TNF-alpha production in KCs of Zucker rats with activation of P38 and JNK. Addition of leptin to normal rats increased LPS-induced hepatic TNF-alpha production in vivo and leptin receptor-deficient Zucker rats showed reduced hepatic TNF-alpha production on addition of LPS in vivo. These findings indicate that P38 and JNK pathways are involved in the signal transduction of leptin enhancement of LPS-induced TNF-alpha production.     

The alpha(4) integrin subunit Tyr(187) has a key role in alpha(4)beta(7)-dependent cell adhesion

The integrin alpha(4)beta(7) is the cell adhesion receptor for the mucosal vascular addressin MAdCAM-1, and this interaction is dominant in lymphocyte homing to Peyer's patch high endothelial venules, and plays key roles in lymphocyte recruitment at sites of inflammation. To identify alpha(4) subunit amino acids important for alpha(4)beta(7)/MAdCAM-1 interaction, we expressed mutant alpha(4) and wild type beta(7) chains in K562 cells and analyzed the effect of the mutations on cell adhesion to a soluble MAdCAM-1 (sMAdCAM-1-Ig). Transfectants expressing mutated alpha(4) at Tyr(187) displayed a substantial decrease in adhesion to this ligand, which was associated with a reduced alpha(4)beta(7)/sMAdCAM-1-Ig interaction, as determined by soluble binding assays. Addition of Mn(2+) to the adhesion assays did not restore the impaired adhesion. Mutations at alpha(4) Gln(152)Asp(153) also affected transfectant adhesion to sMAdCAM-1-Ig, but did not involve an alteration of alpha(4)beta(7)/MAdCAM-1 binding, and adhesion was restored by Mn(2+). Instead, mutations at alpha(4) Asn(123)Glu(124) did not affect this adhesion. Mutation of alpha(4) Tyr(187) abolished alpha(4)beta(7)-mediated cell adhesion to CS-1/fibronectin, an additional ligand for alpha(4)beta(7), while alpha(4) Gln(152)Asp(153) transfectant mutants showed a reduced adhesion. These results identify alpha(4) Tyr(187) as a key residue during receptor alpha(4)beta(7)/ligand interactions, indicating that it plays important roles in alpha(4)beta(7)-mediated leukocyte adhesion, and provide a potential target for therapeutic intervention in several inflammatory pathologies.     

Calcium handling in vasoconstriction to stimulation of alpha 1- and alpha 2-adrenoceptors

Vasoconstriction to stimulation of postsynaptic alpha 1- and alpha 2-adrenoceptors involves different mechanisms of Ca2+ mobilization. Alpha 2-adrenoceptor-mediated vasoconstriction in vivo as well as in vitro is invariably and effectively antagonized by Ca2+ channel blockers, such as nifedipine or verapamil, and is therefore primarily carried by influx of extracellular Ca2+. On the other hand, alpha 1-adrenoceptor stimulation has been linked to both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. The sensitivity of alpha 1-adrenoceptor-mediated vasoconstriction to blockade by Ca2+ channel antagonists depends on how much both mechanisms of Ca2+ mobilization contribute to the contraction process, and varies between vascular tissues and alpha 1-adrenoceptor agonists. The experimental evidence for the differential utilization of Ca2+ in vasoconstriction to alpha 1- and alpha 2-adrenoceptor stimulation is reviewed.     

The role of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle regeneration. Studies in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice.

The role of tumor necrosis factor-alpha (TNF-alpha), an important mediator of the inflammatory response after injury, was investigated in regenerating skeletal muscle. The pattern of expression of TNF-alpha during muscle regeneration was examined by immunohistochemistry in tissue sections of crush-injured or transplanted muscle autografts and in primary cultures of adult skeletal muscle. TNF-alpha was highly expressed in injured myofibers, inflammatory cells, endothelial cells, fibroblasts, and mast cells. Myoblasts and myotubes also expressed TNF-alpha in primary muscle cultures and tissue sections. The essential role of TNF-alpha and its homologue lymphotoxin-alpha (LT-alpha) during muscle regeneration was assessed by basic histology in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice. No difference was apparent in the onset or pattern of muscle regeneration (i.e., inflammatory response, activation and fusion of myoblasts) between the two strains of null mice or between nulls and normal control mice. However, both strains of null mice appeared more prone to bystander damage of host muscle and regeneration distant from the site of injury/transplantation. Although expression of TNF-alpha may play an important role in muscle regeneration, the studies in the null mice show that redundancy within the cytokine system (or some other response) can effectively compensate for the absence of TNF-alpha in vivo.     

Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.     

Neurochemical evidence for two types of presynaptic alpha2-adrenoceptors

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha₂-adrenoceptors through which the release of [³H]noradrenaline and [³H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha₂-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha₂-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha₂-adrenoceptors at the presynaptic axon terminals.Special Issue Dedicated to Dr. Abel Lajtha     

Pigment migration in fish erythrophores is controlled by alpha 2-adrenoceptors

1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha 2-adrenoceptors. 2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha 2-adrenoceptors. 3. The erythrophores themselves were found to possess a K+-sensitive mechanism of aggregation. 4. Some similarities and differences of the alpha 2-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.     

Pigment migration in fish erythrophores is controlled by alpha2-adrenoceptors

• 1.1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha₂-adrenoceptors.
• 2.2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha₂-adrenoceptors.
• 3.3. The erythrophores themselves were found to possess a K⁺-sensitive mechanism of aggregation.
• 4.4. Some similarities and differences of the alpha₂-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.

     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

The development of new triazole based inhibitors of tumor necrosis factor-alpha (TNF-alpha) production

4-Aryl-5-pyridyl and 4-aryl-5-pyrimidyl based inhibitors of TNF-alpha production, which contain a novel triazole 5-member heterocyclic core, are described. Many pyridyl triazoles containing either an alkyl ether or a substituted aryl side chain on the triazole core showed sub-micromolar activity against LPS-induced TNF-alpha, while pyrimidyl triazoles containing an ethoxymethyl side chain exhibited even better inhibitory activity. Secondary screening data are presented for the pyrimidyl triazoles. Triazole 14e combined excellent potency with good oral bioavailability in the rat.     

IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha

IL-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene (MDA-7, IL-24), and AK155 (IL-26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of IL-19. To understand the gene regulation of human IL-19, we identified a human IL-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse IL-19 shared 71% amino acid identity with human IL-19. Treatment of monocytes with mouse IL-19 induced the production of IL-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse IL-19 may play some important roles in inflammatory responses because it up-regulates IL-6 and TNF-alpha and induces apoptosis.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.