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Studies on molecular mechanisms in calcium signaling and cellular energy consumption
KTH, School of Engineering Sciences (SCI), Applied Physics. (Hjalmar Brismar)ORCID iD: 0000-0003-0374-4411
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 76
Keywords [en]
ca2+ signaling, ATP, PercevalHR, NKA, diabetes, Angiotensin
National Category
Medical and Health Sciences
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-204418ISBN: 978-91-7729-337-8 (electronic)OAI: oai:DiVA.org:kth-204418DiVA, id: diva2:1084942
Public defence
2017-04-19, Fire, Scilifelab, Tomtebodavägen 23a, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20170328

Available from: 2017-03-28 Created: 2017-03-27 Last updated: 2017-03-28Bibliographically approved
List of papers
1. Study of energy consumption by Na+/K+ ATPase using PercevalHR
Open this publication in new window or tab >>Study of energy consumption by Na+/K+ ATPase using PercevalHR
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Adenosine triphosphate (ATP) is an important macro molecule that is the prime supplier of energy to run cell metabolism and to regulate several physiological processes in a cell. Despite its central role, the number of non-invasive methods to study ATP on a single cell level in real time are limited. ATPases use energy derived from ATP hydrolysis to maintain cell membrane potential by regulating ion gradients across the plasma membrane. It is generally believed that the Na+/K+-ATPase (NKA) which belongs to the P-type ATPase superfamily represent the main energy consumer among the ATPases. In this study, we set out to quantify ATP consumption by NKA on a single cell level in human embryonic kidney cells (HEK293a) using PercevalHR, a genetically encoded fluorescent biosensor that reports changes in the ATP:ADP during live cell imaging. We demonstrate that ATP hydrolysis by NKA is faster at physiological temperatures (35 -37°C) compared to room temperature. K+ free KREBS pre-treatment increased the ATP consumption by NKA. The inhibition of NKA and SERCA reduced the overall ATP hydrolysis in HEK293a cells demonstrating that these ATPases consume a substantial amount of all ATP produced in the cell. We found that the inhibition of mitochondrial respiration reduced basal ATP:ADP in rat primary proximal tubule cells (PTC) but not in HEK293a cells. In contrast, an inhibition of glycolysis gave a more rapid reduction in basal ATP:ADP in HEK293a compared to PTC, illustrating the Warburg effect in cell lines, where the metabolism has been adapted to a high glucose and low oxygen environment. We demonstrate the use of PercevalHR as a robust tool for studies of dynamic energy consumption by NKA in both cell lines and primary cells. 

Keywords
ATP, PercevalHR
National Category
Medical and Health Sciences
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-204416 (URN)
Note

QC 20170328

Available from: 2017-03-24 Created: 2017-03-24 Last updated: 2017-03-28Bibliographically approved
2. AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
Open this publication in new window or tab >>AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
Show others...
2017 (English)In: BMC Cardiovascular Disorders, ISSN 1471-2261, E-ISSN 1471-2261, Vol. 17, no 1, article id 126Article in journal (Refereed) 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. 

Place, publisher, year, edition, pages
Stockholm: BioMed Central, 2017
Keywords
Calcium, AT1R, Cell imaging, VGCC, hypertension
National Category
Medical and Health Sciences
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-203989 (URN)10.1186/s12872-017-0562-x (DOI)000401701400001 ()28514967 (PubMedID)2-s2.0-85019540748 (Scopus ID)
Funder
Swedish Heart Lung FoundationSwedish Research CouncilMagnus Bergvall FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170328

Available from: 2017-03-21 Created: 2017-03-21 Last updated: 2017-11-29Bibliographically approved
3. Role of TRPM5 channel in insulin secretion from rat β-cells.: TRPM5 and insulin secretion
Open this publication in new window or tab >>Role of TRPM5 channel in insulin secretion from rat β-cells.: TRPM5 and insulin secretion
2014 (English)In: Pancreas, ISSN 0885-3177, E-ISSN 1536-4828, Vol. 43, no 4, p. 597-604Article in journal, Editorial material (Refereed) Published
Abstract [en]

OBJECTIVE:

Several studies have reported that the transient receptor potential melastatin-like subtype 5 (TRPM5) channel, a Ca(2+)-activated monovalent cation channel, is involved in the stimulus-secretion coupling in the mouse pancreatic β-cells. We have studied the role of the TRPM5 channel in regulating insulin secretion and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in the rat β-cells by using triphenylphosphine oxide, a selective inhibitor of the channel.

METHODS:

Insulin secretion from islets from Sprague-Dawley rats was measured in batch incubations. Cytoplasmic free Ca(2+) concentration was measured from single β-cells by fura-2-based microfluorometry.

RESULTS:

Triphenylphosphine oxide did not alter insulin secretion and [Ca(2+)](i) response triggered by KCl or fructose. It inhibited insulin secretion in response to glucose, L-arginine, and glucagon-like peptide 1. It also inhibited glucose-induced insulin secretion by mechanisms that are independent of the adenosine triphosphate-sensitive potassium channels and [Ca(2+)](i) increase.

CONCLUSIONS:

Our results suggest that in the rat islets, TRPM5 is involved in mediating insulin secretion by glucose and l-arginine and in potentiating the glucose-induced insulin secretion by glucagon-like peptide 1.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2014
Keywords
TRPM5, Islets of langerhans, calcium signaling, insulin secretion
National Category
Medical and Health Sciences
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-204559 (URN)
Funder
Swedish Society of Medicine
Note

QC 20170328

Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2017-11-29Bibliographically approved
4. Calcium signaling in a genetically engineered human pancreatic β-cell line
Open this publication in new window or tab >>Calcium signaling in a genetically engineered human pancreatic β-cell line
2015 (English)In: Pancreas, ISSN 0885-3177, E-ISSN 1536-4828, Vol. 44, no 5, p. 773-777Article in journal, Editorial material (Refereed) Published
Abstract [en]

Objectives: The use of primary human β-cells for studying Ca2+ signalingis limited by the scarcity of human pancreatic islets. Rodent insulinomacell lines are widely used, but it is difficult to extrapolate results obtainedfrom rodent cells to human. Recently, a genetically engineered humanβ-cell line EndoC-BH1 has been developed. We have examined whetherthe EndoC-BH1 cells could be used as a model for studying Ca2+ signalingin the β-cells.Methods: We used microscope-based fluorometry to measure cytoplasmicfreeCa2+ concentration from fura-2–loaded single EndoC-BH1 cellscultured on glass cover slips. Ca2+ responses to different agonists of insulinsecretion were studied. Insulin secretion was measured by radioimmunoassay.Results: EndoC-BH1 cells secreted insulin in response to glucose ina dose-dependent manner, and the secretion was enhanced by GLP-1(glucagon-like peptide 1). Glucose, potassium chloride, carbachol, L-arginine,and tolbutamide increased cytoplasmic-free Ca2+ concentration in theEndoC-BH1 cells. We found that GLP-1 was essential for Ca2+ responseto glucose and tolbutamide.Conclusions: We concluded that the EndoC-BH1 cells can be used asmodel cells to study Ca2+ signaling and stimulus-secretion coupling inthe human β-cells.

Keywords
calcium signaling, human β-cell line, insulin, stimulus-secretion coupling, EndoC-BH1 cells
National Category
Medical and Health Sciences
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-204561 (URN)
Note

QC 20170328

Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2017-11-29Bibliographically approved

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