AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

人白介素6受体基因在痘苗病毒载体系统中的表达

人ＩＬ－６受体是一个在各种细胞上广泛表达的跨膜糖蛋白分子,是ＩＬ－６发挥细胞效应所必需的。本文通过将ＩＬ－６ＲｃＤＮＡ重组到痘苗病毒的ＴＫ基因中构建成重组痘苗病毒ＶＩＬ６Ｒ。细胞原位杂交和ＡＰＡＡＰ染色结果表明,感染ＶＩＬ６Ｒ后的Ｖｅｒｏ细胞中,ＩＬ－６Ｒ在ｍＲＮＡ和蛋白水平上均呈现较强的表达。Ｗｅｓｔｅｒｎｂｌｏｔ分析所表达的分子量为８０ｋＤ,表明所表达的产物是糖基化的。ＩＬ－６结合试验表明,表达的膜ＩＬ－６Ｒ能够结合ｒＩＬ－６,说明它是有功能的。利用ＶＩＬ６Ｒ免疫小鼠后,能够刺激较强的抗体产生。从而为进一步研究ＩＬ－６Ｒ的信号传导和构效关系提供了基础。     

Lysophosphatidylcholine potentiates Ca2+ influx, pore formation and p44/42 MAP kinase phosphorylation mediated by P2X7 receptor activation in mouse microglial cells

The P2X7 receptor (P2X7R) is an ATP-gated ion channel highly expressed in microglia. P2X7R plays important roles in inflammatory responses in the brain. However, little is known about the mechanisms regulating its functions in microglia. Lysophosphatidylcholine (LPC), an inflammatory phospholipid that promotes microglial activation, may have some relevance to P2X7R signaling in terms of microglial function. In this study, we examined its effects on P2X7R signaling in a mouse microglial cell line (MG6) and primary microglia. LPC facilitated the sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through P2X7R channels activated by ATP or BzATP. The potentiated increase in [Ca(2+)](i) was actually inhibited by P2X7R antagonists, brilliant blue G and oxidized ATP. The potentiating effect of LPC was not observed with P2Y receptor systems, which are also expressed in MG6 cells. G2A, a receptor for LPC, was expressed in MG6 cells, but not involved in the facilitating effect of LPC on the P2X7R-mediated change in [Ca(2+)](i). Furthermore, LPC enhanced the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase. These results suggest that LPC may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R.     

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4)

The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.