- Publikační typ
- abstrakt z konference MeSH
Large number of extracellular signals is received by plasma membrane receptors which, upon activation, transduce information into the target cell interior via trimeric G-proteins (GPCRs) and induce activation or inhibition of adenylyl cyclase enzyme activity (AC). Receptors for opioid drugs such as morphine (micro-OR, delta-OR and kappa-OR) belong to rhodopsin family of GPCRs. Our recent results indicated a specific up-regulation of AC I (8-fold) and AC II (2.5-fold) in plasma membranes (PM) isolated from rat brain cortex exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Increase of ACI and ACII represented the specific effect as the amount of ACIII-ACIX, prototypical PM marker Na, K-ATPase and trimeric G-protein alpha and beta subunits was unchanged. The up-regulation of ACI and ACII faded away after 20 days since the last dose of morphine. Proteomic analysis of these PM indicated that the brain cortex of morphine-treated animals cannot be regarded as being adapted to this drug because significant up-regulation of proteins functionally related to oxidative stress and alteration of brain energy metabolism occurred. The number of delta-OR was increased 2-fold and their sensitivity to monovalent cations was altered. Characterization of delta-OR-G-protein coupling in model HEK293 cell line indicated high ability of lithium to support affinity of delta-OR response to agonist stimulation. Our studies of PM structure and function in context with desensitization of GPCRs action were extended by data indicating participation of cholesterol-enriched membrane domains in agonist-specific internalization of delta-OR. In HEK293 cells stably expressing delta-OR-G(i)1alpha fusion protein, depletion of PM cholesterol was associated with the decrease in affinity of G-protein response to agonist stimulation, whereas maximum response was unchanged. Hydrophobic interior of isolated PM became more "fluid", chaotically organized and accessible to water molecules. Validity of this conclusion was supported by the analysis of an immediate PM environment of cholesterol molecules in living delta-OR-G(i)1alpha-HEK293 cells by fluorescent probes 22- and 25-NBD-cholesterol. The alteration of plasma membrane structure by cholesterol depletion made the membrane more hydrated. Understanding of the positive and negative feedback regulatory loops among different OR-initiated signaling cascades (micro-, delta-, and kappa-OR) is crucial for understanding of the long-term mechanisms of drug addiction as the decrease in functional activity of micro-OR may be compensated by increase of delta-OR and/or kappa-OR signaling.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- fluidita membrány fyziologie MeSH
- HEK293 buňky MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové lipidy metabolismus MeSH
- mozková kůra metabolismus ultrastruktura MeSH
- opioidní analgetika metabolismus MeSH
- receptory opiátové metabolismus MeSH
- signální transdukce fyziologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.
Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.
- MeSH
- buněčná membrána enzymologie metabolismus MeSH
- down regulace MeSH
- heterotrimerní G-proteiny chemie metabolismus MeSH
- hormony metabolismus MeSH
- křečci praví MeSH
- krysa rodu rattus MeSH
- mozek metabolismus MeSH
- multimerizace proteinu MeSH
- proteiny vázající GTP chemie metabolismus MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- signální transdukce fyziologie MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.
- MeSH
- angiotensin II farmakologie MeSH
- buněčná membrána metabolismus účinky léků MeSH
- časové faktory MeSH
- cholesterol analýza metabolismus MeSH
- financování organizované MeSH
- hormon uvolňující thyreotropin farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 biosyntéza genetika účinky léků MeSH
- receptory thyroliberinu biosyntéza účinky léků MeSH
- teplota MeSH
- transfekce MeSH
- vápník farmakologie metabolismus MeSH
- vápníková signalizace fyziologie účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- buněčné linie MeSH
- cholesterol fyziologie MeSH
- financování organizované MeSH
- guanosin 5'-O-(3-thiotrifosfát) metabolismus MeSH
- hormon uvolňující thyreotropin metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- membránové mikrodomény metabolismus MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus MeSH
- receptory thyroliberinu metabolismus MeSH
- signální transdukce MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
