Enhanced purinoceptor-mediated Ca2+ signalling in L-fibroblasts overexpressing type 1 inositol 1,4,5-trisphosphate receptors

Davis, R. J.; Challiss, R. A. J.; Nahorski, Stefan R.

doi:10.1042/bj3410813

Cited by 12 publications

(2 citation statements)

References 36 publications

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“…In preliminary experiments, however, we found that expressing GFP-IP 3 R1-N affected the [Ca 2+ ] i pattern in both HeLa and COS-7 cells elicited by lower concentrations of ATP than used in the present study (results not shown). Stable overexpression of IP 3 R has been reported to have a rather complex effect on [Ca 2+ ] i signalling patterns, depending on the cells and systems used [22][23][24][25]. To the best of our knowledge, detailed analysis of transient overexpression of IP 3 R on [Ca 2+ ] i signalling patterns has never been reported.…”

Section: Effect Of Tagging Ip 3 R1 With Gfpmentioning

confidence: 99%

The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

Nakayama

Hattori

Uchida

et al. 2004

Biochemical Journal

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The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca(2+) channel protein that plays crucial roles in generating complex Ca(2+) signalling patterns. IP(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP(3)-generating agonist ATP and Ca(2+) leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca(2+) concentration. Interestingly, store-operated (-like) Ca(2+) entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca(2+) stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca(2+). We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca(2+) concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca(2+) stores.

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Section: Effect Of Tagging Ip 3 R1 With Gfpmentioning

confidence: 99%

The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

Nakayama

Hattori

Uchida

et al. 2004

Biochemical Journal

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“…P2YRs are seven membrane‐spanning, G‐protein‐coupled receptors numbering to a total of eight (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ). Their activation triggers generation of inositol 1,4,5‐trisphosphate, Ca 2+ mobilization from intracellular stores and, for some subtypes, adenylate cyclase stimulation (Davis et al, 1999; Homolya et al, 1999). P2XRs, numbering to a total of seven (P2X 1–7 ), are ligand‐gated plasma membrane channels directly activated by extracellular ATP, in the absence of generation of additional second messengers besides monovalent and divalent ion fluxes.…”

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confidence: 99%

Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Solini

Chiozzi²,

Morelli³

et al. 2003

Journal Cellular Physiology

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Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.

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Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells

et al. 2005

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Enhanced purinoceptor-mediated Ca2+ signalling in L-fibroblasts overexpressing type 1 inositol 1,4,5-trisphosphate receptors

Cited by 12 publications

References 36 publications

The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells

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