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AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0003-0374-4411
Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk,.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sverige.ORCID-id: 0000-0003-0578-4003
Visa övriga samt affilieringar
2017 (Engelska)Ingår i: BMC Cardiovascular Disorders, ISSN 1471-2261, E-ISSN 1471-2261, Vol. 17, nr 1, artikel-id 126Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background: Blockers of angiotensin II type 1   receptor (AT 1 R) and the voltage gated calcium channel 1.2 (Ca V 1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT 1 R signaling and whether there is a reciprocal regulation of AT 1 R signaling by Ca V 1.2.

Methods: To elucidate these questions, we have studied the Ca 2+  signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca 2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of Ca V 1.2 blockers.  Semi- quantitative imaging methods were used to assess the plasma membrane expression of AT 1 R and G-protein activation.

Results: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca 2+  response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca 2+  response. The up-regulation of the Ca 2+  response to repeated 1 nM AngII doses and the down- egulation of the Ca 2+  response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT 1 R plasma membrane expression. The Ca 2+  response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the Ca V 1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT 1 R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT 1 R is sensitive to calcium influx.

Conclusions: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca 2+  channel blockers as mono-therapy in hypertension. 

Ort, förlag, år, upplaga, sidor
Stockholm: BioMed Central, 2017. Vol. 17, nr 1, artikel-id 126
Nyckelord [en]
Calcium, AT1R, Cell imaging, VGCC, hypertension
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Medicinsk teknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-203989DOI: 10.1186/s12872-017-0562-xISI: 000401701400001PubMedID: 28514967Scopus ID: 2-s2.0-85019540748OAI: oai:DiVA.org:kth-203989DiVA, id: diva2:1083618
Forskningsfinansiär
Hjärt-LungfondenVetenskapsrådetMagnus Bergvalls StiftelseScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20170328

Tillgänglig från: 2017-03-21 Skapad: 2017-03-21 Senast uppdaterad: 2017-11-29Bibliografiskt granskad
Ingår i avhandling
1. Studies on molecular mechanisms in calcium signaling and cellular energy consumption
Öppna denna publikation i ny flik eller fönster >>Studies on molecular mechanisms in calcium signaling and cellular energy consumption
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Ion signaling plays fundamental role in cell survival. Na+ and Ca2+ are critical players in ion signaling. Cells spend the major amount of energy to maintain and regulate Na+ and Ca2+ gradients across the cell membrane. Any disruption in cellular energy consumption by plasma membrane ATPases affects ion signaling and vice versa. This thesis is a combination of four separate research studies. In the first study, We measured ATP consumption dynamics of Na+/K+-ATPase using a genetically encoded fluorescent indicator called Perceval HR. we demonstrate that PercevalHR is an excellent tool to visualize ATP:ADP in mammalian cells.

In the second study, We studied the role of calcium signaling and TRP channels in angiotensin II type 1 receptor  signaling cascade. We prove that low inhibition of CaV1.2 with physiological and therapeutically relevant concentration of Angiotensin II up regulate AT1R signaling.

In the third study, We studied the role of the TRPM5 channel in regulating insulin secretion, and cytoplasmic free calcium concentration in the rat β-cells by usingtriphenyl phosphine oxide, a selective inhibitor of the channel.

In the fourth study, We tested whether, the genetically engineered human β-cell line (EndoC-BH1) could be used as models for studying Ca2+signaling in the context of Type II Diabetes. We found that the EndoC-BH1 cells could be a relevant model to study stimulus-secretion coupling and Ca2+ signaling in the human β-cells.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2017. s. 76
Nyckelord
ca2+ signaling, ATP, PercevalHR, NKA, diabetes, Angiotensin
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Biologisk fysik
Identifikatorer
urn:nbn:se:kth:diva-204418 (URN)978-91-7729-337-8 (ISBN)
Disputation
2017-04-19, Fire, Scilifelab, Tomtebodavägen 23a, Solna, 09:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20170328

Tillgänglig från: 2017-03-28 Skapad: 2017-03-27 Senast uppdaterad: 2017-03-28Bibliografiskt granskad
2. Quantitative bioimaging in single cell signaling
Öppna denna publikation i ny flik eller fönster >>Quantitative bioimaging in single cell signaling
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.

Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.

Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.

In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.

Abstract [sv]

Avbildning av biologiska prover har i flera hundra år varit ett sätt för forskare att undersöka biologiska system. Med utvecklingen av immunofluoresens inmärkn-ing och fluoresens-mikroskopi förbättrades de viktigaste aspekterna av mikroskopi,kontrast och specificitet. Sedan 1941 har vi sett kontinuerligt mer mångsidigt och frekvent användning av fluorosense-mikroskopi i biologisk forskning.

Jon-mikroskopi har länge varit en metod att studera signalering i cell-system. Genom användning av fluorosenta jon-sensorer går det att mäta variationer avjon koncentrationer i levande celler som resultat av yttre påverkan. Genom att använda Ca2+ mikroskopi har jag visat att det finns en omvänd koppling mellan kalcium-kanaler i plasma-membran och angiotensin II typ 1 receptorn (ett proteininvolverat i blodtrycksreglering). Detta har direkta implikationer för behandlingav högt blodtryck, en av de mer vanliga sjukdomarna i västvärlden idag med överen miljard drabbade patienter i världen 2016.

Efter detta projekt vidgades mitt fokus till att inkludera superupplösnings-mikroskopi. Denna avhandling inkluderar ett arbete fokuserat på tolkningen av superupplösnings-mikroskopi data från neuronal Na+, K+ - ATPase α3, en jon-pump som återställer cellernas jonbalans i samband med cell signalering. Mikroskopi-datan korreleras mot elektrofysiologi experiment och modeller för att illustrera möjliga artefakter i superupplösnings-mikroskopi som måste tas i beaktande i samband med tolkning av data.

Jag fortsatte med att utveckla mjukvara för analys av data från singel-molekyl-lokalisations-mikroskopi där fokuset för mjukvaran framförallt varit på användarvänligheten. Detta då jag hoppas att den kommer vara användbar för ett bredare forskingsfält. Mjukvaran användes även i ett separat projekt för att identifiera överuttrycks-artefakter i transfekterade celler.

I det avslutande arbetet använder jag superupplösnings-mikroskopi för att karakterisera de tidiga stegen i mitokondriell apoptos. Jag identifierar när och var i cellen de olika proteinerna involverade i apoptos signaleringen är aktiverade och interagerar.

Ort, förlag, år, upplaga, sidor
Kungliga Tekniska högskolan, 2017. s. 52
Serie
TRITA-FYS, ISSN 0280-316X ; 64
Nyckelord
Super resolution imaging, fluoresence, bioimaging, cells, FRET, cluster analysis, labeling, image analysis.
Nationell ämneskategori
Biofysik
Identifikatorer
urn:nbn:se:kth:diva-215076 (URN)978-91-7729-546-4 (ISBN)
Disputation
2017-10-27, Air and Fire, Science for Life Laboratory, Tomtebodavägen 23a, Solna, 09:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20171003

Tillgänglig från: 2017-10-03 Skapad: 2017-10-01 Senast uppdaterad: 2017-10-03Bibliografiskt granskad

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