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Lysophosphatidic acid: Physiological effects and structure-activity relationships
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lipids havepreviously been considered primarily as building blocks of the cell membrane, but are now also recognized as important cell signaling molecules. Lysophosphatidic acid (LPA) is a glycerophospholipid consisting of a phosphate head group, a linker region, and a lipophilic tail. LPA has earlier been shown to exert a diversity of cellular effects such as aggregation, apoptosis, contraction, migration, and proliferation. The effects of LPA are elicited by activation of its cognate G protein-coupled receptors LPA1, LPA2, and LPA3. In the present study we have used cultures of human smooth muscle cells (SMCs) and erythroleukemia cells (HEL), and isolated human platelets to characterize physiological effects of LPA compared with adrenaline and noradrenaline as well as structure-activity relationships of LPA. SMCs were isolated from biopsies of human myometrium obtained at cesarean sections. We show that cultured myometrial SMCs express multiple LPA and α2-adrenergic receptor subtypes. Treatment of SMCs with LPA and noradrenaline resulted in increases in proliferation. However, LPA elicits a much more pronounced stimulatory effect than noradrenaline. The ability to increase calcium might be one explanation why LPA is more effective. Further studies indicated that several pathways mediate the growth stimulatory effect of LPA where transactivation of epidermal growth factor receptors through matrix metalloproteinases as well as calcium/calmodulin-dependent protein kinases appears to be important. LPA enantiomers and LPA analogues were synthesized and characterized due to their capacity to increase calcium in HEL cells. Our study is the first to show that both natural (R) and unnatural (S) LPA enantiomers are capable of stimulating cells, suggesting LPA receptors are not stereoselective. Moreover, we have synthesized a LPA analogue with higher maximal effect than LPA by reducing the hydrocarbon chain length. In platelets we demonstrated that LPA is a weak calciumelevating compound which failed to stimulate aggregation. However, in combination with adrenaline, another weak platelet agonist, a complete aggregatory response was obtained in blood from some healthy individuals. These results are important since platelet activation is a key step in distinguishing normal from pathological hemostasis. Since LPA is present at high concentrations in atherosclerotic lesions, the synergistic effect of LPA and adrenaline might be a new risk factor for arterial thrombosis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2002. , 100 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 751
Keyword [en]
Lysophospholipids pharmacology, physiology, structure-activity relationship, muscle, smooth, drug effects, myometrium, norepinephrine, epinephrine, signal transduction
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-5237ISBN: 91-7373-192-7 (print)OAI: oai:DiVA.org:liu-5237DiVA: diva2:21230
Public defence
2002-11-08, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Note
On the day of the public defence the status of the article IV was: Submitted for publication.Available from: 2006-01-17 Created: 2006-01-17 Last updated: 2012-01-25Bibliographically approved
List of papers
1. Different proliferative responses of Gi/o-protein-coupled receptors in human myometrial smooth muscle cells: a possible role of calcium
Open this publication in new window or tab >>Different proliferative responses of Gi/o-protein-coupled receptors in human myometrial smooth muscle cells: a possible role of calcium
1998 (English)In: Journal of Molecular Neuroscience, ISSN 0895-8696, Vol. 11, no 1, 11-21 p.Article in journal (Refereed) Published
Abstract [en]

The majority of studies investigating the proliferative effect of Gi/o-protein-coupled receptor agonists are performed in recombinant receptor systems or cell lines. In these systems the relative stoichiometry of receptors compared to other cell components might be changed, which may lead to anomalies in cellular responses in contrast to natural occurring systems. In the present study, we have used primary cultures of smooth muscle cells (SMCs) isolated from human myometrium to characterize the proliferative effects of agonists binding to two different G protein-coupled receptors. Treatment of quiescent SMCs with lysophosphatidic acid (LPA) and noradrenaline resulted in significant increases in [3H]thymidine incorporation. However, LPA was almost four times more effective than noradrenaline in this respect. The proliferative effects of the agonists could be completely blocked by pertussis toxin, indicating that the response are mediated through Gi/o-proteins. The selective α2-adrenergic receptor (α2-AR) antagonist yohimbine dose-dependently reduced the effect of noradrenaline suggesting that the proliferative response was mediated through α2-ARs. The proliferative effects induced by LPA and noradrenaline was markedly reduced in SMCs treated with the tyrosine kinase inhibitor genistein and the cAMP elevating compound forskolin. However, LPA but not noradrenaline induced rapid rises in the cytosolic free Ca2+ concentration [Ca2+]i. The ability to increase Ca2+ might be one explanation why LPA produce a more pronounced proliferative response than noradrenaline in primary cultures of human myometrial SMCs.

Keyword
Uterus, smooth muscle cells, G protein-coupled receptors, DNA synthesis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13736 (URN)10.1385/JMN:11:1:11 (DOI)
Available from: 2006-01-17 Created: 2006-01-17 Last updated: 2009-08-20
2. Inhibition of Ca2 +/calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells
Open this publication in new window or tab >>Inhibition of Ca2 +/calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells
2003 (English)In: Cell Biology International, ISSN 1065-6995, Vol. 27, no 4, 341-347 p.Article in journal (Refereed) Published
Abstract [en]

Human myometrial smooth muscle cells (SMCs) were used to evaluate the proliferative activity of lysophosphatidic acid (LPA). This study specifically focuses on the role of Ca2+/calmodulin-dependent protein (CaM) kinase and epidermal growth factor (EGF) receptor tyrosine kinase. Myometrial SMCs were cultured from biopsies taken at Cesarean sections. The expression of LPA receptors was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and DNA-synthesis was measured by [3H]thymidine incorporation. LPA1, LPA2, and LPA3receptor subtypes were detected in the SMCs using RT-PCR. KN-62, an inhibitor of CaM kinase, and Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, dose-dependently decreased LPA-stimulated [3H]thymidine incorporation. Furthermore, BB-3103, an inhibitor of matrix metalloproteinases (MMPs), also reduced DNA-synthesis induced by LPA in these cells. The results show, for the first time, that human myometrial SMCs express all three known LPA receptor subtypes. Growth stimulatory effects of LPA on myometrial SMCs seems to be mediated by several pathways, where transactivation of EGF receptors through MMPs appears to be of importance. Furthermore, CaM kinase activity may be critical for LPA signaling since inhibition of CaM kinase totally abolish the proliferative effect of LPA.

Keyword
Calcium, Calmodulin, DNA-synthesis, Epidermal growth factor, Lysophosphatidic acid, Smooth muscle cells
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13737 (URN)10.1016/S1065-6995(02)00352-9 (DOI)
Available from: 2006-01-17 Created: 2006-01-17
3. Synergistic activation of human platelets by adrenaline and lysophosphatidic acid
Open this publication in new window or tab >>Synergistic activation of human platelets by adrenaline and lysophosphatidic acid
2002 (English)In: Haematologica, ISSN 0390-6078, Vol. 87, no 7, 730-739 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND AND OBJECTIVES: Platelet reactivity is regulated by various important bioactive and physiologic substances. The objective of this study was to characterize lysophosphatidic acid (LPA)-triggered responses in human platelets. In addition, the effect of LPA was compared with that of other activators and possible synergistic interactions were evaluated.

DESIGN AND METHODS: LPA-triggered cytosolic Ca(2+) responses were measured using fura-2-loaded platelets in a spectrofluorometer. Furthermore, platelet aggregation and secretion were analyzed in a lumi-aggregometer and protein tyrosine phosphorylation was detected with the Western blot technique.

RESULTS: LPA dose-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in platelets. This response involved both influx of extracellular Ca(2+) and release of Ca(2+) from intracellular stores. However, in comparison with other platelet agonists, i.e. thrombin and adenosine 5'-diphosphate (ADP), LPA was a very weak Ca(2+)-elevating agent. Furthermore, we observed that the LPA-induced rise in [Ca(2+)](i) was markedly suppressed by cyclic nucleotide-elevating agents. In functional studies, LPA failed to stimulate platelet aggregation and secretion. However, in combination with adrenaline, another weak platelet agonist, LPA could induce an irreversible and complete aggregatory response. There was an individual variation in aggregatory response and tyrosine phosphorylation when LPA and adrenaline were combined. These agents induced a powerful response on platelets from some individuals, but had a weak or no effect on others.

INTERPRETATION AND CONCLUSIONS: The present study shows, for the first time, that isolated platelets from some healthy blood donors respond synergistically to a combination of LPA and adrenaline. Platelet activation is a key step in distinguishing normal hemostasis from pathologic hemostasis. Increased knowledge about this mechanism might help to predict individual responses and provide new insights into molecular mechanisms responsible for pathologic thrombosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13738 (URN)
Available from: 2006-01-17 Created: 2006-01-17 Last updated: 2009-05-28
4. Lack of stereospecificity in lysophosphatidic acid enantiomerinduced calcium mobilization in human erythroleukemia cells
Open this publication in new window or tab >>Lack of stereospecificity in lysophosphatidic acid enantiomerinduced calcium mobilization in human erythroleukemia cells
Show others...
2003 (English)In: Lipids, ISSN 0024-4201, Vol. 38, no 10, 1057-1064 p.Article in journal (Refereed) Published
Abstract [en]

Lysophosphatidic acid (LPA) is a lipid mediator that, among several other cellular responses, can stimulate cells to mobilize calcium (Ca2+). LPA is known to activate at least three different subtypes of G protein-coupled receptors. These receptors can then stimulate different kinds of G proteins. In the present study, LPA and LPA analogs were synthesized from (R)- and (S)-glycidol and used to characterize the ability to stimulate Ca2+ mobilization. The cytosolic Ca2+ concentration ([Ca2+]i) was measured in fura-2-acetoxymethylester-loaded human erythroleukemia (HEL) cells. Furthermore, a reverse transcriptase polymerase chain reaction was used to characterize LPA receptor subtypes expressed in HEL cells. The results show that HEL cells mainly express LPA1 and LPA2, although LPA3 might possibly be expressed as well. Moreover, LPA and its analogs concentration-dependently increased [Ca2+]i in HEL cells. The response involved both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. This is the first time the unnatural (S)-enantiomer of LPA, (S)-3-O-oleoyl-1-O-phosphoryl-glycerol, has been synthesized and studied according to its ability to activate cells. The results indicate that this group of receptors does not discriminate between (R)- and (S)-enantiomers of LPA and its analogs. When comparing ether analogs having different hydrocarbon chain lengths, the tetradecyl analog (14 carbons) was found to be the most effective in increasing [Ca2+]i. Pertussis toxin treatment of the HEL cells resulted in an even more efficient Ca2+ mobilization stimulated by LPA and its analogs. Furthermore, at repeated incubation with the same ligand no further increase in [Ca2+]i was obtained. When combining LPA with the ether analogs no suppression of the new Ca2+ signal occurred. All these findings may be of significance in the process of searching for specific agonists and antagonists of the LPA receptor subtypes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13739 (URN)
Available from: 2006-01-17 Created: 2006-01-17

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