Nicotinic receptors (NRs) play an important role in the cholinergic regulation of heart functions, and converging evidence suggests a diverse repertoire of NR subunits in the heart. A recent hypothesis about the plasticity of β NR subunits suggests that β2-subunits and β4-subunits may substitute for each other. In our study, we assessed the hypothetical β-subunit interchangeability in the heart at the level of mRNA. Using two mutant mice strains lacking β2 or β4 NR subunits, we examined the relative expression of NR subunits and other key cholinergic molecules. We investigated the physiology of isolated hearts perfused by Langendorff's method at basal conditions and after cholinergic and/or adrenergic stimulation. Lack of β2 NR subunit was accompanied with decreased relative expression of β4-subunits and α3-subunits. No other cholinergic changes were observed at the level of mRNA, except for increased M3 and decreased M4 muscarinic receptors. Isolated hearts lacking β2 NR subunit showed different dynamics in heart rate response to indirect cholinergic stimulation. In hearts lacking β4 NR subunit, increased levels of β2-subunits were observed together with decreased mRNA for acetylcholine-synthetizing enzyme and M1 and M4 muscarinic receptors. Changes in the expression levels in β4-/- hearts were associated with increased basal heart rate and impaired response to a high dose of acetylcholine upon adrenergic stimulation. In support of the proposed plasticity of cardiac NRs, our results confirmed subunit-dependent compensatory changes to missing cardiac NRs subunits with consequences on isolated heart physiology.NEW & NOTEWORTHY In the present study, we observed an increase in mRNA levels of the β2 NR subunit in β4-/- hearts but not vice versa, thus supporting the hypothesis of β NR subunit plasticity that depends on the specific type of missing β-subunit. This was accompanied with specific cholinergic adaptations. Nevertheless, isolated hearts of β4-/- mice showed increased basal heart rate and a higher sensitivity to a high dose of acetylcholine upon adrenergic stimulation.
- MeSH
- acetylcholin farmakologie MeSH
- antagonisté muskarinových receptorů farmakologie MeSH
- atropin farmakologie MeSH
- cholinesterasové inhibitory farmakologie MeSH
- hexamethonium farmakologie MeSH
- isoprenalin farmakologie MeSH
- myokard metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- neostigmin farmakologie MeSH
- nikotinové receptory metabolismus MeSH
- srdce účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.
- MeSH
- acetylcholin metabolismus MeSH
- acetylcholinesterasa analýza metabolismus MeSH
- butyrylcholinesterasa analýza metabolismus MeSH
- GPI-vázané proteiny analýza metabolismus MeSH
- izoenzymy analýza metabolismus MeSH
- kolagen analýza metabolismus MeSH
- krysa rodu rattus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- membránové proteiny analýza metabolismus MeSH
- myokard enzymologie MeSH
- potkani Wistar MeSH
- proteiny nervové tkáně analýza metabolismus MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- butyrylcholinesterasa * fyziologie imunologie izolace a purifikace krev MeSH
- ELISA * MeSH
- enzymatické testy MeSH
- hydrolýza MeSH
- kinetika MeSH
- lidé MeSH
- protilátky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Acetylcholínesteráza (AChE) a butyrylcholínesteráza (BChE) tvoria malú rodinu enzýmov nazývaných cholínesterázy. Tieto enzýmy sú v organizme buď solubilné, alebo kotvené prostredníctvom kotviacich proteínov kolagénu Q (ColQ) a membránovej kotvy bohatej na prolín (PRiMA). Poznatky z molekulovej biológie a genetiky cholínesteráz a ich kotviacich proteínov viedli ku príprave mutantných myší s absenciou rôznych molekulových foriem cholínesteráz. Do dnešných dní bolo pripravených viacero mutantných myší s genetickou modifikáciou na úrovni génov pre cholínestrázy alebo ich kotviace proteíny. Medzi myši s mutáciou v génoch kódujúcich cholínesterázy patria predovšetkým: myši s celkovou absenciou AChE, myši s celkovou absenciou BChE, myši s deléciou exónu 5 a 6 v géne kódujúcom AChE a myši s absenciou AChE vo svaloch. Medzi myši s mutáciou v génoch kódujúcich kotviace proteíny cholínesteráz patria: myši s absenciou AChE a BChE kotvenej prostredníctvom proteínu ColQ a myši s absenciou AChE a BChE kotvenej prostredníctvom proteínu PRiMA. Na základe štúdia zmien vyplývajúcich z absencie cholínesteráz, tieto mutantné zvieratá výrazne prispeli k obohateniu doterajších poznatkov o cholínesterázach a cholinergickom nervovom systéme.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) represent a small family of enzymes called cholinesterases. These enzymes are in the organisms either soluble or anchored through anchoring proteins collagen Q (ColQ) and proline-rich membrane anchor (PRiMA). Knowledge of molecular biology and genetics of cholinesterase and their anchoring proteins resulted in the preparation of mutant mice with the absence of different molecular forms of cholinesterases. So far a number of mutant mice were prepared with a genetic modification on the genes encoding cholinesterases or anchoring proteins. The mice with mutation in the genes encoding the cholinesterases are: the mice with the absence of AChE, mice with the absence of BChE, mice with a deletion of exon 5 and 6 in the AChE gene and mice with the absence of AChE in muscles. The mice with a mutation in the genes encoding anchoring proteins include the mice with the absence of AChE and BChE anchored by ColQ and mice with the absence of AChE and BChE anchored by PRiMA. The study of adaptation changes results from the absence of cholinesterases led to the enrichment of existing knowledge about cholinesterases and the cholinergic nervous system.
- MeSH
- acetylcholinesterasa * genetika krev metabolismus MeSH
- butyrylcholinesterasa genetika krev metabolismus MeSH
- cholinesterasy * genetika krev metabolismus MeSH
- delece genu MeSH
- genetická transkripce MeSH
- kolagen genetika metabolismus MeSH
- modely nemocí na zvířatech MeSH
- mutantní kmeny myší MeSH
- svalové proteiny genetika metabolismus MeSH
- změny tělesné hmotnosti MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by a central cholinergic deficit. Non-neuronal cholinergic changes are, however, described as well. Here we focused on possible changes in the activity of the plasma cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), in hospitalized AD patients. We analyzed plasma AChE and BChE activities with regards to age, gender, body mass index (BMI), cognitive functions, and ability to perform activities of daily living in AD patients in comparison to healthy subjects. We observed lower AChE activity and trend toward lower BChE activity in AD patients, which both correlated with low BMI. AD patients unable to perform basic activities of daily living (feeding, bathing, dressing, and grooming) showed reduced plasma AChE activities, while worse spatial orientation was linked to lower BChE activities. Three out of four AD patients with the lowest BChE activities died within one year. In conclusion, progressed AD was accompanied by lower plasma AChE activity and trend toward lower BChE activity, which correlated with BMI and deficits in different components of the AD.
- MeSH
- acetylcholinesterasa krev MeSH
- Alzheimerova nemoc patofyziologie terapie MeSH
- biochemická analýza krve MeSH
- butyrylcholinesterasa krev MeSH
- činnosti denního života * MeSH
- hospitalizace MeSH
- index tělesné hmotnosti * MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- změna polohy v prostoru fyziologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- pH detekční metody, histochemické metody,
- MeSH
- acetylcholinesterasa * farmakologie fyziologie nedostatek škodlivé účinky MeSH
- biosenzitivní techniky metody normy přístrojové vybavení MeSH
- butyrylcholinesterasa * farmakologie fyziologie nedostatek škodlivé účinky MeSH
- chemické techniky analytické * využití MeSH
- cholinesterasové inhibitory MeSH
- cholinesterasy farmakologie fyziologie MeSH
- imunohistochemie metody normy přístrojové vybavení MeSH
- kolorimetrie metody normy přístrojové vybavení trendy MeSH
- lidé MeSH
- radiometrie metody normy přístrojové vybavení využití MeSH
- spektrofotometrie metody normy přístrojové vybavení MeSH
- titrace metody normy přístrojové vybavení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Cholinergický systém v srdci môže byť buď neuronálneho, alebo neneuronálneho pôvodu. Neuronálny cholinergický systém v srdci je reprezentovaný pregangliovými parasympatikovými dráhami, parasympatikovými intrakardiálnymi gangliami a postgangliovými parasympatikovými neurónmi, ktoré projektujú predovšetkým do oblastí predsiení, SA uzla a AV uzla. Neneuronálny cholinergický systém je tvorený samotnými kardiomyocytmi, ktoré majú kompletnú výbavu pre syntézu a vylučovanie acetylcholínu. Podľa súčasných poznatkov neneuronálny cholinergický systém v srdci má vplyv na reguláciu srdcovej činnosti v priebehu aktivácie sympatiku a zohráva úlohu aj pri energetickom metabolizme kardiomyocytov. Acetylcholín či už neuronálneho alebo neneuronálneho pôvodu pôsobí v srdci prostredníctvom muskarínových a nikotínových receptorov. Účinok acetylcholínu v srdci je ukončovaný prostredníctvom cholínesteráz, acetylcholínesterázy a butyrylcholínesterázy. V poslednej dobe čoraz viac odborných publikácii naznačuje, že zvýšenie cholinergického tonusu v srdci prostredníctvom inhibítorov cholínesteráz má pozitívny vplyv pri niektorých ochoreniach kardiovaskulárneho systému, ako je napríklad zlyhávajúce srdce. Z toho dôvodu by sa inhibítory cholínesteráz mohli v budúcnosti úspešne používať pri terapii niektorých ochorení kardiovaskulárneho systému.
The cholinergic system of the heart can be either of neuronal or non-neuronal origin. The neuronal cholinergic system in the heart is represented by preganglionic parasympathetic pathways, intracardiac parasympathetic ganglia and postganglionic parasympathetic neurons projecting to the atria, SA node and AV node. The non-neuronal cholinergic system consists of cardiomyocytes that have complete equipment for synthesis and secretion of acetylcholine. Current knowledge suggests that the non-neuronal cholinergic system in the heart affects the regulation of the heart during sympathetic activation. The non-neuronal cholinergic system of the heart plays also a role in the energy metabolism of cardimyocites. Acetylcholine of both neuronal and non-neuronal origin acts in the heart through muscarinic and nicotinic receptors. The effect of acetylcholine in the heart is terminated by cholinesterases acetylcholinesterase and butyrylcholinesterase. Recently, papers suggest that the increased cholinergic tone in the heart by cholinesterase inhibitors has a positive effect on some cardiovascular disorders such as heart failure. For this reason, the cholinesterase inhibitors might be used in the treatment of certain cardiovascular disorders in the future. Key words: cholinergic system • heart innervation • non-neuronal cholinergic system of the heart • receptors • cholinesterases in the heart
- MeSH
- acetylcholin sekrece MeSH
- acetylcholinesterasa fyziologie metabolismus MeSH
- butyrylcholinesterasa fyziologie metabolismus MeSH
- cholin-O-acetyltransferasa fyziologie MeSH
- cholinergní vlákna fyziologie MeSH
- cholinesterasové inhibitory farmakologie terapeutické užití MeSH
- cholinesterasy fyziologie MeSH
- ganglia parasympatická fyziologie MeSH
- kardiomyocyty fyziologie MeSH
- lidé MeSH
- neurony cholinergní * fyziologie MeSH
- nikotinové receptory fyziologie MeSH
- parasympatická vlákna postgangliová fyziologie MeSH
- parasympatický nervový systém * fyziologie MeSH
- receptory muskarinové fyziologie MeSH
- srdce * inervace MeSH
- srdeční selhání farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
