Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.
- MeSH
- adenosintrifosfát chemie MeSH
- adenylátcyklasový toxin metabolismus MeSH
- AMP cyklický biosyntéza MeSH
- antigeny CD11b genetika metabolismus MeSH
- antigeny CD18 genetika metabolismus MeSH
- biologický transport fyziologie MeSH
- Bordetella pertussis metabolismus MeSH
- buněčné linie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- fagocyty metabolismus MeSH
- lidé MeSH
- makrofágový antigen 1 biosyntéza genetika metabolismus MeSH
- signální transdukce fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Muscarinic M(2) receptors preferentially couple with the G(i/o) class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M(2) receptors (CHO-M(2) cells) whether direct interaction of M(2) receptors with G(s) and G(q/11) G-proteins is responsible for the latter effects. Suppression of the G(s)alpha subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M(2) cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC(50) of 79 microM. Removal of the G(q),G(11), or both alpha subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G(s)alpha subunit had no effect. Similar results obtained in CHO cells expressing M(1) receptors that preferentially couple with G(s) and G(q/11) G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M(2) receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M(2) receptor coupling with the alpha subunits of G(s) and G(q/11) G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.
- MeSH
- AMP cyklický biosyntéza MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- financování organizované MeSH
- inositolfosfáty metabolismus MeSH
- křečci praví MeSH
- malá interferující RNA farmakologie MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 fyziologie MeSH
- proteiny vázající GTP - alfa-podjednotky Gs fyziologie MeSH
- receptor muskarinový M2 fyziologie MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.
- MeSH
- acetylcholin metabolismus MeSH
- acetylcholinesterasa genetika MeSH
- adrenergní receptory biosyntéza MeSH
- AMP cyklický biosyntéza MeSH
- down regulace MeSH
- financování organizované MeSH
- fosfatidylinositoly metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- imunoanalýza MeSH
- myši knockoutované MeSH
- myši MeSH
- plíce metabolismus MeSH
- proteiny vázající GTP metabolismus MeSH
- radioligandová zkouška MeSH
- receptory muskarinové biosyntéza MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.
- MeSH
- AMP cyklický biosyntéza MeSH
- financování organizované MeSH
- folikulární buňky cytologie metabolismus účinky léků MeSH
- folikuly stimulující hormon farmakologie MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- glukuronosyltransferasa genetika metabolismus MeSH
- insulinu podobný růstový faktor I farmakologie MeSH
- kultivované buňky MeSH
- kyselina hyaluronová biosyntéza metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 metabolismus MeSH
- oocyty metabolismus účinky léků MeSH
- prasata MeSH
- proliferace buněk účinky léků MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- AMP cyklický biosyntéza MeSH
- bodová mutace MeSH
- dítě MeSH
- fibrózní dysplazie monostotická etiologie patologie terapie MeSH
- kyčelní kloub patologie MeSH
- lidé MeSH
- mladiství MeSH
- předškolní dítě MeSH
- receptory buněčného povrchu genetika MeSH
- skolióza etiologie patologie terapie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- přehledy MeSH
