Publications by authors named "Nour B Bishara"

4 Publications

  • Page 1 of 1

Cytochrome P450 products and arachidonic acid-induced, non-store-operated, Ca2+ entry in cultured bovine endothelial cells.

Endothelium 2005 Jul-Aug;12(4):153-61

Microvascular Biology Group, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia.

Endothelial cells possess multiple mechanisms for the control of Ca2+ influx during agonist and mechanical stimulation. Increased intracellular Ca2+ during such events is important in the production of vasoactive substances including NO, prostacyclin, and, possibly, endothelium-derived hyperpolarizing factor(s). The present studies examined the effect of arachidonic acid on cellular Ca2+ entry and the underlying mechanisms by which this fatty acid regulates entry. Studies were conducted in cultured bovine aortic endothelial cells (passages 3 to 6) with changes in intracellular Ca2+ determined using the fluorescent Ca2+-sensitive indicator fura 2. Arachidonic acid (1 to 50 microM) stimulated Ca2+ entry from the superfusate without affecting Ca2+ release from intracellular stores. 2-aminoethoxydiphenyl borate (2APB) (100 microM) added at the peak of Ca2+ entry did not inhibit arachidonic acid-induced Ca2+ entry but, in contrast, significantly inhibited entry stimulated by ATP (1 microM). Arachidonic acid-induced Ca2+ entry was inhibited by econazole (1 microM), but not indomethacin (10 microM) or nordihydroguairetic acid (10 microM), suggesting the involvement of cytochrome P450 monooxygenase metabolite of arachidonic acid. Oleic acid (10 microM) was ineffective in inducing Ca2+ entry, whereas linoleic acid (10 microM) stimulated Ca2+ entry but by a mechanism insensitive to econazole. Collectively the data demonstrate that primary cultured aortic endothelial cells possess a Ca2+ entry mechanism modulated by arachidonic acid. This mode of Ca2+ entry appears to operate independently of store depletion-mediated mechanisms.
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http://dx.doi.org/10.1080/10623320500227036DOI Listing
December 2005

Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.

Clin Exp Pharmacol Physiol 2003 Sep;30(9):653-8

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.
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http://dx.doi.org/10.1046/j.1440-1681.2003.03892.xDOI Listing
September 2003

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

J Cell Physiol 2002 Oct;193(1):80-92

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
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http://dx.doi.org/10.1002/jcp.10153DOI Listing
October 2002

Capacitative Ca(2+) entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C.

Br J Pharmacol 2002 Jan;135(1):119-28

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

1. Agonists increase endothelial cell intracellular Ca(2+), in part, by capacitative entry, which is triggered by the filling state of intracellular Ca(2+) stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca(2+) stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP(3) receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP(3) receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca(2+) entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3. 2APB (30 - 300 microM) inhibited Ca(2+) entry induced by both agonists (ATP 1 microM, bradykinin 0.1 microM) and receptor-independent mechanisms (thapsigargin 1 microM, ionomycin 0.5 and 5 microM). 2APB did not diminish endothelial cell ATP-induced production of IP(3) nor effect in vitro binding of [(3)H]-IP(3) to an adrenal cortex binding protein. Capacitative Ca(2+) entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca(2+) release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3 - 10 microM) inhibited ATP-induced Ca(2+) release and capacitative Ca(2+) entry. Further, xestospongin C inhibited capacitative Ca(2+) entry induced by thapsigargin (1 microM) and ionomycin (0.5 microM). 5. The data are consistent with a mechanism of capacitative Ca(2+) entry in vascular endothelial cells which requires (a) IP(3) receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca(2+) release and entry pathways.
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http://dx.doi.org/10.1038/sj.bjp.0704465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573124PMC
January 2002