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INSTITUTE OF PHYSIOLOGY CAS

Cutting-edge science for health

Publications

Signalling bias as the way to side-effect-free medication (22.7. 2020)

More than 30 % of currently marketed medications act via G-protein coupled receptors (GPCRs). This type of membrane receptors is involved in the control of a wide range of physiological processes, from the processing of sensory stimuli, through the regulation of behavior and mood, hormonal and immune responses, to the control of autonomic functions and cell proliferation. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists biased towards a given signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. Enormous research has been done on agonists biased towards either G-protein or arrestin mediated pathway.

In our laboratory we focus on the study of muscarinic acetylcholine receptors, which belong to the typical representatives of GPCR. Here, as a proof of concept, we demonstrate unprecedented signalling bias solely at the level of G‑protein-mediated signalling. We present agonists of muscarinic acetylcholine receptors, a member of GPCR family, that exclusively inhibit cAMP synthesis through activation of Gi‑protein pathway and thus are functionally selective for M2 and M4 receptor subtypes. Muscarinic receptors M2, M4 represent one of the pharmacological targets in the treatment of pain. Newly discovered agonists may lead to the development of new non-addictive analgesics such as opiates, or immunity-weakening as steroid analgesics. Such muscarinic analgesics would not cause side effects mediated by activation of the Gq protein signaling pathway, such as incontinence, excessive salivation and sweating, and more.

A) General scheme of coupling GPCRs with individual subtypes of G‑proteins or arrestins after activation by a traditional full agonist (left) and an agonist preferring Gi‑protein activation (right). AC, adenylyl cyclase; PLC, phospholipase C. B) Activation of M2 receptor by the traditional agonist (blue) resulting in stimulation of the Gi and Gs‑protein pathway leading to a decrease in cAMP level in the cell at low concentration of agonist and an increase in cAMP level at high concentrations of the agonist. Activation of M2 receptor by two different Gi‑biased agonists (red, orange) resulting in stimulation of the Gi‑protein pathway only and decrease in cAMP level.

Randáková A, Nelic D, Ungerová D, Nwokoye P, Su Q, Doležal V, El-Fakahany EE, Boulos J, Jakubík J. Novel M2-Selective, Gi-Biased Agonists of Muscarinic Acetylcholine Receptors. Br J Pharmacol. 2020 May;177(9):2073-2089. doi: 10.1111/bph.14970.

Lipokine 5-PAHSA is Regulated by Adipose Triglyceride Lipase and Primes Adipocytes for de novo Lipogenesis in Mice (22.7. 2020)

5-PAHSA belongs to newly discovered lipids which show a great promise as anti-diabetic and anti-inflammatory compounds, but their action is poorly explored. Animal and cell culture experiments showed that 5-PAHSA helps to import glucose into the cells in a different way from insulin. While insulin stimulates lipid accumulation, administration of 5-PAHSA results in the activation of energy-demanding metabolic processes of fatty acid synthesis. Therefore, glucose is used more efficiently.

The effect of a bioactive lipid 5-PAHSA on glucose and lipid metabolism. Labeling of metabolic pathways in mice and advanced analytical methods were used to explore the beneficial effect of 5-PAHSA bioactive lipids in glucose utilization.

Lipokine 5-PAHSA is Regulated by Adipose Triglyceride Lipase and Primes Adipocytes for de novo Lipogenesis in Mice. Paluchova V, Oseeva M, Brezinova M, Cajka T, Bardova K, Adamcova K, Zacek P, Brejchova K, Balas L, Chodounska H, Kudova E, Schreiber R, Zechner R, Durand T, Rossmeisl M, Abumrad NA, Kopecky J, Kuda O. Diabetes. 2019 Dec 5. pii: db190494. doi: 10.2337/db19-0494.

Elucidation of TRPA1 activation mechanisms may help to understand the pathophysiology of chronic pain (3.2. 2020)

A plethora of serious diseases is followed by chronic pain the molecular mechanisms of which are not fully unraveled. The research on TRPA1 ion channel, which is involved in these mechanisms, is conducted by the researchers from the Laboratory of Cellular Neurophysiology of the Institute of Physiology CAS. In collaboration with the laboratory CBMN from the University of Bordeaux they proved that the activity of human TRPA1 is significantly regulated by membrane phospholipids at negative membrane potentials. They succeeded in identifying the binding site of TRPA1 channel, which interacts with phospholipids at low concentrations of calcium ions. At higher concentrations of calcium ions, the same region represents the binding site for a regulatory protein calmodulin, which modulates the TRPA1 channel in an activity-dependent manner. These new findings suggest that the correct phospholipid composition of the plasma membrane is important for TRPA1 activation under physiological conditions. Membrane phospholipids help to stabilize the interface between TRPA1 subunits to maintain the transduction of signals into the channel opening.

 

(A) Electrophysiological recordings of TRPA1 channel‘s responses to carvacrol (100 µM) and to elevated concentration of calcium ions (from 0 to 0.5 mM). Recordings from two F11 cells recorded with a pipette filled with control solution (left) and solution with the specific peptide T1003-P1034 added (right). Lower, there is a suggested mechanism of competition between calmodulin (CaM) and phospholipid (PIP2) for the binding site of TRPA1. (B) The binding site for phospholipids in the region of two neighboring subunits of TRPA1 channel. Only the transmembrane part of the protein, which contributes to the binding and transduction of signals into the channel gate, is shown (PDB code of the structure 6pqq). The role of phenylalanine Phe1020 in the interaction was revealed in the study.

MACIKOVA L, SINICA V, KADKOVA A, VILLETTE S, CIACCAFAVA A, FAHERTY J, LECOMTE S, ALVES ID, VLACHOVA V: Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials. The FEBS Journal 286: 3664-3683, 2019.

Innovative method of the Forhkead transcription factor FOXO3 regulation (29.1. 2020)

The study published in prestigious journal eLife identifies small molecule compounds that interact with the Forkhead box O3 transcription factor (FOXO3) and modulate its activity.

FOXO3, with the characteristic fork head DNA-binding domain is part of the O subclass of the forkhead family of transcription factors. These transcription factors have important roles in mammalian cells in regards to regulating cell homeostasis, differentiation, longevity and steer cell death. The activity of FOXO3 in particular contributes to therapy-resistance programs that protect cancer cells during chemo and radiotherapy. Recent studies have also found the DNA-binding domain (DBD) of FOXO aid protein-protein interactions with other key regulators of longevity and death, and drug resistance. A reversible inhibition of FOXO3 activity by small compounds thereby might boost anti-tumor immune responses.

In order to inhibit the FOXO3 activity, it was first necessary to identify small molecule compounds that could block the interaction between FOXO3 and DNA. Using the structural data of FOXO3 DBD and FOXO4 DBD, the researchers developed six different pharmacophore models that were used for in silico screening of small molecule compound databases. Selected compounds were then tested for their ability to inhibit FOXO3 function both in vitro, and in cancer cell lines. The interactions of these compounds with FOXO3 DBD were assessed using NMR spectroscopy and docking studies.

The teams of Dr. V. Obšilová from the Institute of Physiology CAS in BIOCEV and prof. T. Obšil from the Faculty of Science, Charles University with the team of prof. M. J. Ausserlechner from the Medical University of Innsbruck, Austria identified the compounds S9 and its oxalate salt S9OX as compounds able to inhibit the FOXO3 activity in cancer cells. They also proposed that due to their mode of binding to FOXO3 DBD, these compounds may also interfere with protein-protein interactions of FOXO3. The advantage of these compounds is the strict control of application-dose and -time and the fact that they are not immunogenic allowing repeated applications – so dose- and application time can be adjusted to damage cancer cells or boost anti-cancer immunity, but also limit unwanted side effects of FOXO-inhibition on stem cells and other somatic tissues. Future research will be focused on investigation whether or not S9 can be used as a chemical foundation for developing FOXO-regulatory compounds that would control the functions and target gene subsets of FOXO transcription factors.

Compounds S9 blocks the DNA binding surface of Forkhead transcription factor FOXO3. The figure shows the structural model of the DNA-binding domain of FOXO3 with bound compound S9 based on data from NMR measurements and docking simulations.

Hagenbuchner, J.+, Obšilová, V.+, Kaserer, T.+, Kaiser, N., Rass, B., Pšenáková, K., Dočekal, V., Alblová, M., Kohoutová, K., Schuster, D., Aneichyk, T., Veselý, J., Obexer, P., Obšil, T.*, Ausserlechner, M. J.* Modulating FOXO3 transcriptional activity by small, DBD-binding molecules. eLife. 2019, 8(Dec 4), e48876.  doi: 10.7554/eLife.48876

Transcription factor Hif-1a is required for the correct development of the sympathetic nervous system and innervation of the heart (16.1. 2020)

Hypoxia-inducible factor 1 (Hif-1) is the master regulator of transcriptional responses of cells to decreased oxygen availability. Research teams of the Institutes of Biotechnology and Physiology CAS and the 1st Medical Faculty UK showed that genetic mutation of the Hif-1a gene suppresses the embryonic development of preganglionic and postganglionic neurons of the sympathetic nerve system and negatively affects sympathetic innervation of the heart that plays a primary role in the regulation of heart rate and contractility. Mice with conditional deletion of Hif-1a gene exhibited coronary artery anomalies and decreased cardiac contractile function. These data indicate that deregulation of the transcription factor Hif-1a can result in serious cardiovascular diseases associated with the autonomic nervous system dysbalance and open the way to a development of new therapeutic strategies.

Impaired sympathetic innervation of hearts with Hif-1a deletion (Hif1aCKO): Immunohistochemical staining of tyrosine hydroxylase (TH) in posterior view of hearts, and quantification of TH-positive fibers per ventricle area in E16.5 control and Hif1aCKO littermates.

Bohuslavová, Romana - Čerychová, Radka - Papoušek, František - Olejníčková, Veronika - Bartoš, M. - Goerlach, A. - Kolář, František - Sedmera, David - Semenza, G.L. - Pavlínková, Gabriela: HIF-1 alpha is required for development of the sympathetic nervous system. Proceedings of the National Academy of Sciences of the United States of America. Roč. 116, č. 27 (2019), s. 13414-13423. ISSN 0027-8424, IF: 9.580, 2018. DOI: 10.1073/pnas.1903510116

The resistance of pancreatic beta cells to oxidative stress, which accompanies diabetes type 2, can be increased by mitochondria-targeted antioxidants (15.1. 2020)

Increased blood glucose induces secretion of the hormone insulin in β cells of Langerhans islets in the pancreas. his response is regulated by redox signalization, where a slight increase of reactive oxygen species (ROS) serves as a messenger for the regulation of protein pathways leading to the secretion of insulin from insulin granules in β cells. On the contrary, excessive production of ROS causes pathological oxidative stress which accompanies many diseases such as diabetes type 2. Oxidative stress can be suppressed by enhancing antioxidant defense.  Mitochondria are important energy factories of the cells and are also one of the main sources of ROS. In the present paper, we tested the effect of new antioxidant molecules, namely SkQ, S3QEL and S1QEL, which target the sites of ROS production in mitochondria. We uncovered the detailed mechanism of their effect in various sites of mitochondria, where they specifically prevent ROS formation and thus show antioxidant role. However, they can also have pro-oxidative properties. This is dependent on energy metabolism of the cell and thus substrate availability (for example glucose). As an example, SkQ antioxidant shows an antioxidant property when a cell has excessive energy substrate supply which happens in vivo after a meal, i.e. postprandial. If there is a shortage of energy substrate, happening in vivo during fasting, SkQ behaves in short-term pro-oxidatively and can even increase already established oxidative stress. Detailed knowledge of the activity of these selected antioxidant in pancreatic β cells can be used in diabetes treatment.

Figure of possible SkQ activity: Suggested mechanisms for antioxidant action of matrix-targeted antioxidant SkQ: (a) antioxidative two-electron reduction of SkQ to SkQH2 plus regeneration (oxidation of SkQH2) within the mitochondrial electron transport complex I, based on reference [1]; (b) antioxidative two-electron reduction of SkQ to SkQH2 within complex III and regeneration at the complex I, based on reference [1]

Plecitá-Hlavatá; Lydie - Engstová; Hana - Ježek; Jan - Holendová; Blanka - Tauber; Jan - Petrásková; Lucie - Křen; Vladimír - Ježek; Petr . Potential of Mitochondria-Targeted Antioxidants to Prevent Oxidative Stress in Pancreatic beta-cells . Oxidative Medicine and Cellular Longevity. 2019; 2019(2019)); 1826303 . IF = 4.868.

A promising pharmacological approach in cardiovascular disease prevention (9.7. 2019)

Epoxyeicosatrienoic acids (EETs), cytochrome P450 epoxygenase metabolites of arachidonic acid, represent a promising pharmacological approach in cardiovascular disease prevention. In our study, cardioprotective effects of a novel, stable and orally active agonistic 14,15-EET analog EET-B on the progression of post-ischemic heart failure  was examined in spontaneously hypertensive rats (SHR), a pre-clinical rodent model of human essential hypertension. SHR were subjected to myocardial infarction and the effects of continuous EET-B treatment before and after MI on post-ischemic left ventricular function, myocardial fibrosis and inflammation were analyzed. As EET-based therapies can attenuate the progression of HF by mechanisms involving activation of heme oxygenase-1, its immunopositivity in viable myocytes of the ischemic myocardium was also determined. We demonstrated that EET-B treatment improved post-ischemic left ventricular function, markedly increased heme oxygenase-1 immunopositivity in cardiomyocytes subjected myocardial infarction and reduced cardiac inflammation and fibrosis. These findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce remodeling in SHR subjected to myocardial infarction.

8.Neckář, Jan - Khan, M. A. H. - Gross, G. J. - Cyprová, Michaela - Hrdlička, Jaroslav - Kvasilová, A. - Falck, J. R. - Campbell, W. B. - Sedláková, Lenka - Škutová, Šárka - Olejníčková, Veronika - Gregorovičová, Martina - Sedmera, David - Kolář, František - Imig, J. D. Epoxyeicosatrienoic acid analog EET-B attenuates post-myocardial infarction remodeling in spontaneously hypertensive rats. Clinical science. Roč. 133, č. 8 (2019), s. 939-951. ISSN 0143-5221, IF: 5,237 (2018).

Small amounts of inorganic nitrate or beetroot provide substantial protection from salt-induced Increases in blood pressure (4.4. 2019)

Adding tiny amounts of beetroot or dietary nitrate to salty food products might help prevent high blood pressure, according to a preliminary study of rats.

To reduce the risk of salt-induced hypertension, medical authorities have emphasized dietary guidelines promoting high intakes of potassium and low intakes of salt that provide molar ratios of potassium to salt of ≥1:1. However, during the past several decades, relatively few people have changed their eating habits sufficiently to reach the recommended dietary goals for salt and potassium. Thus, new strategies that reduce the risk of salt-induced hypertension without requiring major changes in dietary habits would be of considerable medical interest. In the current studies in a widely used model of salt-induced hypertension, the Dahl salt-sensitive rat, we found that supplemental dietary sodium nitrate confers substantial protection from initiation of salt-induced hypertension when the molar ratio of added nitrate to added salt is only ≈1:170. Provision of a low molar ratio of added nitrate to added salt of ≈1:110 by supplementing the diet with beetroot also conferred substantial protection against salt-induced increases in blood pressure. The results suggest that on a molar basis and a weight basis, dietary nitrate may be ≈100× more potent than dietary potassium with respect to providing substantial resistance to the pressor effects of increased salt intake. Given that leafy green and root vegetables contain large amounts of inorganic nitrate, these findings raise the possibility that fortification of salty food products with small amounts of a nitrate-rich vegetable concentrate may provide a simple method for reducing risk for salt-induced hypertension.

Effects of supplemental sodium nitrate or beetroot on salt-induced increases in blood pressure. A, Time course of 24-h averages of systolicarterial pressure. B, Time course of 24-h averages of diastolic arterial pressure. C, Mean changes in systolic arterial pressure induced by salt loading. D, Mean changes in diastolic arterial pressure induced by salt loading. *P < 0.05.

Morris Jr, R. Curtis - Pravenec, Michal - Šilhavý, Jan - DiCarlo, E. Stephen - Kurtz, W. Theodore: Small amounts of inorganic nitrate or beetroot provide substantial protection from salt-induced increases in blood pressure. Hypertension. Roč. 73, 2019. doi: 10.1161/HYPERTENSIONAHA.118.12234. [Epub ahead of print]. ISSN 0194-911X, IF: 6.857.

The effect of gut microbiome on expression of genes involved in regulation of stress response in chronic stress (11.2. 2019)

The scientists from the Institutes of Physiology and Microbiology CAS have shown, that absence of gut microbiota leads to altered behavior in social conflict and changes of expression of genes involved in regulation of stress response in chronically stressed mice.

Mammals are naturally colonized by microorganisms, commonly referred as the microbiome. Substantial fraction of the microbiome represent bacteria. Some bacteria are capable of synthetizing chemical substances, such as hormones or neurotransmitters, and thus influence the host both on local and whole-organism level. Disruption of the microbiome is associated with several immune and neuropsychiatric disorders and stress may disrupt the balance between the host and the microbiome. When facing potential danger, stress response acts towards maintaining the homeostasis by activating variety of mechanisms such as sympatho-adrenal system or the hypothalamo-pituitary-adrenal (HPA) axis. Acute stress is generally not harmful, however when prolonged or poorly managed, it can lead to serious side effects.

For studying the effects of microbiome on the host the germ-free mice are used as animal model. It is known that microbiome alters the behavior and can alter humoral response to acute stress. In collaboration with our colleagues from the Institute of Microbiology CAS we have focused on the effects of chronic psychosocial stress in germ-free mice. Our results have shown that the absence of microbiome affects the murine behavior in social conflicts (Fig. 1) and modulates the expression of genes involved in regulation of stress response in adrenal gland (Fig. 2), local paracrine signalization in colon and expression of cytokines in mesentery lymph nodes and colon. This study contributes to understanding the crosstalk between microbiota and the host brain, especially during chronic stress.

Fig. 1. Time spent in individual defensive behavioral patterns (upright posture, freezing and escape/flight) and in total defensive behavior (B) of specific pathogen-free (SPF) and germ-free (GF) mice. Data are expressed as the means ± SEM; *P < 0.05.

Fig. 2. Responses of genes encoding adrenal steroidogenesis, catecholamine biogenesis and glucocorticoid metabolism following social defeat in specific pathogen-free (SPF) and germ-free (GF) mice. MC2R, melanocortin 2 receptor; StAR, steroidogenic acute regulatory protein; Cyp11a1, cholesterol side-chain cleavage enzyme; TH, tyrosine hydroxylase; PNMT, phenylethanolamine N-methyltransferase; 11HSD1 and 11HSD2, 11b-hydroxysteroid dehydrogenase type 1 and type 2. The data are expressed as the means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Vodička, Martin - Ergang, Peter - Hrnčíř, Tomáš - Mikulecká, Anna - Kvapilová, Pavlína - Vagnerová, Karla - Šestáková, Blanka - Fajstová, Alena - Hermanová, Petra - Hudcovic, Tomáš - Kozáková, Hana - Pácha, Jiří: Microbiota affects the expression of genes involved in HPA axis regulation and local metabolism of glucocorticoids in chronic psychosocial stress. Brain Behavior and Immunity. Roč. 73, Oct 2018 (2018), s. 615-624. ISSN 0889-1591, IF: 6.306, 2017.

Alignment of mother and offspring body clock could prevent diseases such as heart disease and obesity (9.1. 2019)

The scientists from the Institute of Physiology CAS found out that the care provided by a mother can impact the body clock and health of offspring after birth. By reducing abnormalities in the body clock of offspring, it may be possible to develop therapies for serious lifestyle-related diseases, such as heart disease and obesity.

The body has an internal clock that regulates sleepiness over a 24 hour period, called your circadian rhythm. The circadian system is important so that processes in our body are synchronised with day and night, i.e. when it is light or dark outside. Disturbances in these mechanisms can lead to poor health, such as heart disease.

The mother-offspring interaction is very important for health later in adulthood. This is the first study to provide compelling evidence that the circadian characteristics of a mother may positively affect the likelihood of disease developing in offspring. Providing better maternal care significantly reduced abnormalities in the circadian system and resulted in a lower likelihood of development of heart disease.

The study conducted by the Institute of Physiology of the Czech Academy of Sciences was performed in two strains of laboratory rats, in which the maternal care and synchrony of their circadian clocks with external day/night cycle differed. The effect of maternal care provided by the genetic mother of these pups was compared with maternal care of the foster mother. The pups either had an aberrant circadian system and were genetically determined to develop disease in adulthood or they were healthy controls. The researchers tested the effect of maternal care on the body clock before and just after weaning, and on their activity rhythms, heart rate and blood pressure in adulthood. Proper maternal care provided to pups genetically predisposed to develop disease led to improvement of their clock function and abolished the rise in their heart rate in adulthood.

The data obtained in pups before weaning were based on population samples because the researchers could not assess circadian rhythms of the clocks in each individual pup within the body without disturbing maternal behaviour. Additionally, in the rat strain spontaneously developing disease, the molecular mechanisms connecting the circadian clock and the pathology is not understood.

These results point to a real possibility to reduce abnormalities in the offspring’s body clock and therefore limit the progression of disease in order to improve health. The future research will be directed at understanding in more detail how an aberrant circadian system contributes to the progression of disease. This research is worth future explorations as it may provide novel therapies for serious life-style related diseases in humans.

Olejníková, Lucie - Polidarová, Lenka - Behuliak, Michal - Sládek, Martin - Sumová, Alena: Circadian alignment in a foster mother improves the offspring's pathological phenotype.  Journal of Physiology. Roč. 596, č. 23 (2018), s. 5757-5775. ISSN 0022-3751, IF: 4.540, 2017.

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