1 Agonists increase endothelial cell intracellular Ca 2+ , in part, by capacitative entry, which is triggered by the ®lling 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 a ects 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 e ects 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 mM) inhibited Ca 2+ entry induced by both agonists (ATP 1 mM, bradykinin 0.1 mM) and receptor-independent mechanisms (thapsigargin 1 mM, ionomycin 0.5 and 5 mM). 2APB did not diminish endothelial cell ATP-induced production of IP 3 nor e ect 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 una ected. 4 Similarly to 2APB, xestospongin C (3 ± 10 mM) inhibited ATP-induced Ca 2+ release and capacitative Ca 2+ entry. Further, xestospongin C inhibited capacitative Ca 2+ entry induced by thapsigargin (1 mM) and ionomycin (0.5 mM). 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.
Hyperglycemia is a major risk factor for endothelial dysfunction and vascular disease, and in the current study, the link to glucose-induced abnormal intracellular Ca2+ (Cai2+) homeostasis was explored in bovine aortic endothelial cells in high glucose (HG; 25 mmol/l) versus low glucose (LG; 5.5 mmol/l; control). Transient receptor potential 1 (TRPC1) ion channel protein, but not TRPC3, TRPC4, or TRPC6 expression, was significantly increased in HG versus LG at 72 h. HG for 4, 24, and 72 h did not change basal Cai2+ or ATP-induced Cai2+ release; however, the amplitude of sustained Cai2+ was significantly increased at 24 and 72 h and reduced by low concentration of the putative, but nonspecific, TRPC blockers, gadolinium, SKF-96365, and 2-aminoethoxydiphenyl borate. Treatment with TRPC1 antisense significantly reduced TRPC1 protein expression and ATP-induced Ca2+ entry in bovine aortic endothelial cells. Although the link between HG-induced changes in TRPC1 expression, enhanced Ca2+ entry, and endothelial dysfunction require further study, the current data are suggestive that targeting these pathways may reduce the impact of HG on endothelial function.
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.
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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