scholarly journals Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

2006 ◽  
Vol 190 (2) ◽  
pp. 373-384 ◽  
Author(s):  
Shannon M Gifford ◽  
Fu-Xian Yi ◽  
Ian M Bird
Keyword(s):  
Endothelial Cells ◽  
Uterine Artery ◽  
Purinergic Receptor ◽  
Cell Types ◽  
P2y Receptors ◽  
Receptor Expression ◽  
Receptor Subtype ◽  
Initial Transient ◽  
Receptor Coupling ◽  
Specific Variation

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca2+ concentration ([Ca2+]i), but the P-UAEC show a heightened sustained phase. In order to establish whether thiswas due to an altered subclass of purinergic receptor (P2), both the dose dependencyof [Ca2+]i responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca2+ signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca2+ signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca2+is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca2+ entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca2+]i response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca2+signaling.

scholarly journals Pregnancy-enhanced store-operated Ca2+ channel function in uterine artery endothelial cells is associated with enhanced agonist-specific transient receptor potential channel 3-inositol 1,4,5-trisphosphate receptor 2 interaction

2006 ◽  
Vol 190 (2) ◽  
pp. 385-395 ◽  
Author(s):  
Shannon M Gifford ◽  
Fu-Xian Yi ◽  
Ian M Bird
Keyword(s):  
Endothelial Cells ◽  
Uterine Artery ◽  
Transient Receptor Potential ◽  
Purinergic Receptor ◽  
Receptor Potential ◽  
Cell Types ◽  
Transient Receptor Potential Channels ◽  
Significant Difference ◽  
Transient Receptor ◽  
Trisphosphate Receptor

We have previously shown that endothelial cells (EC) derived from the uterine artery (UA) of both pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes show a biphasic intracellular free Ca2+ ([Ca2+]i) response after ATP stimulation. In each case, the initial transient peak, caused by the release of Ca2+ from the intracellular Ca2+ stores, is mediated by purinergic receptor-Y2 and is very similar in both cell types. However, the sustained phase in particular, caused by the influx of extracellular Ca2+, is heightened in the P-UAEC, and associates with an increased ability of the cells to demonstrate enhanced capacitative Ca2+ entry (CCE) via store-operated channels (SOCs). Herein we demonstrated that the difference in the sustained [Ca2+]i response is maintained for at least 30 min. When 2-aminoethoxydiphenyl borate (2APB) (an inhibitor of the inosital 1,4,5-trisphosphate receptor (IP3R) and possibly SOC) was used in conjunction with ATP, it was capable of completely inhibiting CCE. Since 2APB can inhibit SOC in some cell types and 2APB was capable of inhibiting CCE in the UAEC model, the role of SOC in CCE was first evaluated using the classical inhibitor La3+. The ATP-induced sustained phase was inhibited by 10 μM La3+, implying a role for SOC in the [Ca2+]i response. Since canonical transient receptor potential channels (TRPCs) have recently been identified as putative SOCs in many cell types, including EC, the expression levels of several isoforms were evaluated in UAEC. Expression of TRPC3 and TRPC6 channels in particular was detected, but no significant difference in expression level was found between NP- and P-UAEC. Nonetheless, we were able to show that IP3R2 interacts with TRPC3 in UAEC, forming a protein complex, and that this interaction is considerably enhanced in an agonist sensitive manner by pregnancy. Thus, while IP3R and TRPC isoforms are not altered in their expression by pregnancy, enhanced functional interaction of TRPC3 with IP3R2 may underlie pregnancy-enhanced CCE in the UAEC model and so explain the prolonged [Ca2+]i sustained phase seen in response to ATP.

scholarly journals Microglia and Neuroinflammation: What Place for P2RY12?

2021 ◽  
Vol 22 (4) ◽  
pp. 1636
Author(s):  
Albert Gómez Morillas ◽  
Valérie C. Besson ◽  
Dominique Lerouet
Keyword(s):  
Purinergic Receptor ◽  
P2y Receptors ◽  
Receptor Expression ◽  
Brain Cells ◽  
Physiological Functions ◽  
Genetic Tools ◽  
And Migration ◽  
Activation Status

Microglia are immune brain cells involved in neuroinflammation. They express a lot of proteins on their surface such as receptors that can be activated by mediators released in the microglial environment. Among these receptors, purinergic receptor expression could be modified depending on the activation status of microglia. In this review, we focus on P2Y receptors and more specifically on P2RY12 that is involved in microglial motility and migration, the first step of neuroinflammation process. We describe the purinergic receptor families, P2RY12 structure, expression and physiological functions. The pharmacological and genetic tools for studying this receptor are detailed thereafter. Last but not least, we report the contribution of microglial P2RY12 to neuroinflammation in acute and chronic brain pathologies in order to better understand P2RY12 microglial role.

scholarly journals Multifunctional Liposomes Modulate Purinergic Receptor-Induced Calcium Wave in Cerebral Microvascular Endothelial Cells and Astrocytes: New Insights for Alzheimer’s disease

2021 ◽  
Author(s):  
Greta Forcaia ◽  
Beatrice Formicola ◽  
Giulia Terribile ◽  
Sharon Negri ◽  
Dmitry Lim ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Purinergic Receptor ◽  
P2y Receptors ◽  
Microvascular Endothelial Cells ◽  
Cultured Astrocytes ◽  
Intracellular Ca ◽  
Microvascular Endothelial

AbstractIn light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).

scholarly journals Diabetes-related sex differences in the brain endothelin system following ischemia in vivo and in human brain endothelial cells in vitro

2020 ◽  
Vol 98 (9) ◽  
pp. 587-595 ◽  
Author(s):  
Yasir Abdul ◽  
Weiguo Li ◽  
Juan D. Vargas ◽  
Emily Grant ◽  
Lianying He ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sex Differences ◽  
Cell Types ◽  
Diabetic Rats ◽  
Receptor Subtype ◽  
Endothelin System ◽  
Eta Receptor ◽  
The Brain

The endothelin (ET) system has been implicated to contribute to the pathophysiology of cognitive impairment and stroke in experimental diabetes. Our goals were to test the hypotheses that (1) circulating and (or) periinfarct ET-1 levels are elevated after stroke in both sexes and this increase is greater in diabetes, (2) ET receptors are differentially regulated in the diabetic brain, (3) brain microvascular endothelial cells (BMVEC) of female and male origin express the ETA receptor subtype, and (4) diabetes- and stroke-mimicking conditions increase ET-1 levels in BMVECs of both sexes. Control and diabetic rats were randomized to sham or stroke surgery. BMVECs of male (hBEC5i) and female (hCMEC/D3) origin, cultured under normal and diabetes-mimicking conditions, were exposed to normoxia or hypoxia. Circulating ET-1 levels were higher in diabetic animals and this was more pronounced in the male cohort. Stroke did not further increase plasma ET-1. Tissue ET-1 levels were increased after stroke only in males, whereas periinfarct ET-1 increased in both control and diabetic females. Male BMVECs secreted more ET-1 than female cells and hypoxia increased ET-1 levels in both cell types. There was sexually dimorphic regulation of ET receptors in both tissue and cell culture samples. There are sex differences in the stroke- and diabetes-mediated changes in the brain ET system at the endothelial and tissue levels.

Regulation of P2X7nucleotide receptor function in human monocytes by extracellular ions and receptor density

2001 ◽  
Vol 280 (4) ◽  
pp. C943-C953 ◽  
Author(s):  
Lalitha Gudipaty ◽  
Benjamin D. Humphreys ◽  
Gary Buell ◽  
George R. Dubyak
Keyword(s):  
Cell Surface ◽  
Human Blood ◽  
Pore Formation ◽  
Cell Types ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Blood Monocytes

P2X receptors function as ATP-gated cation channels. The P2X7receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X7receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X7receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+and Cl−with K+and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30–100 μM ATP was sufficient for activation of nonselective pores by P2X7receptors. Comparison of P2X7receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X7receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X7receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+and Cl−. These mechanisms may prevent adventitious P2X7receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

2006 ◽  
Vol 290 (4) ◽  
pp. H1433-H1445 ◽  
Author(s):  
Jacqueline M. Cale ◽  
Ian M. Bird
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Uterine Artery ◽  
Cell Types ◽  
Enos Activity ◽  
Key Residues ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca2+ and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.

CCK-B receptors produce similar signals but have opposite growth effects in CHO and Swiss 3T3 cells

1997 ◽  
Vol 273 (5) ◽  
pp. C1449-C1457 ◽  
Author(s):  
Katharina Detjen ◽  
David Yule ◽  
Min-Jen Tseng ◽  
John A. Williams ◽  
Craig D. Logsdon
Keyword(s):  
B Cells ◽  
Dna Synthesis ◽  
Cell Types ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Growth Responses ◽  
3T3 Cells ◽  
Growth Effects ◽  
Receptor Coupling ◽  
Swiss 3T3

Rat cholecystokinin-B (CCK-B) receptors were transfected into Chinese hamster ovary (CHO)-K1 (CHO-CCK-B) and Swiss 3T3 (Swiss 3T3-CCK-B) cells, and the effects of receptor activation on cell proliferation and intracellular signaling were investigated. CCK octapeptide (CCK-8) treatment had no effect on cell growth in quiescent CHO-CCK-B cells but inhibited DNA synthesis, proliferation, and colony formation when the cells were grown in fetal bovine serum (FBS). In contrast, CCK-8 stimulated DNA synthesis in quiescent Swiss 3T3-CCK-B cells and had no effect when the cells were grown in FBS. These differences in growth responses were not due to differences in the level of receptor expression, as similar numbers of receptors were present in both cell types. To determine whether the different growth effects were due to differences in receptor coupling to common second messenger pathways, we investigated the effects of CCK-8 on several known intracellular signals. In both cell types, CCK-8 stimulated increases in intracellular Ca2+concentration and polyphosphoinositide hydrolysis with similar potencies and efficacies. CCK-8 also stimulated arachidonate release from both cell types, although the potency was higher in the CHO cells. Adenosine 3′,5′-cyclic monophosphate generation was observed at high agonist concentrations in both cell types and was much greater in cells with higher receptor density. In summary, receptor activation had opposite effects on growth parameters in CHO and Swiss 3T3 cells, but only minor differences were observed in the characteristics of CCK-B receptor coupling to specific second messengers in the two cell types. Thus cellular context is a principal determinant of the biological effects of CCK-B receptor activation, and differences in biological responses may occur independently of major differences in receptor coupling.

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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