ATP caused a dose‐dependent, receptor‐mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P 2 receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca 2+ ‐EGTA saline, suggesting the involvement of intracellular calcium stores. Pre‐treatment of glial cultures with an intracellular Ca 2+ chelator abolished the stimulatory effects of ATP. Thapsigargin (1 µ m ), an inhibitor of Ca 2+ ‐ATPase from the Ca 2+ pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 µ m ), a phospholipase C inhibitor, attenuated the ATP‐stimulatory effect on calcium transients and blocked ATP‐evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP‐induced glutamate release. Furthermore, inhibition of the glutamate transporters p ‐chloromercuri‐phenylsulfonic acid and l trans‐ pyrolidine‐2,4‐dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl − channel blocker, 5‐nitro‐2(3‐phenylpropylamino)‐benzoate, reduced ATP‐induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium‐dependent anion‐transport sensitive mechanism.

EGTA

Thapsigargin

Calcium in biology

10.1046/j.1471-4159.2001.00272.x

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Citations (177)

Author Index Vol. 77, 2003

Neuroendocrinology (2003)

Stephanie M. Krasnow Dawit N. Teklemichael Donald K. Clifton David Wynick Robert A. Steiner

10.1159/000072103

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Echinacea-induced cytosolic Ca2+ elevation in HEK293

BMC Complementary and Alternative Medicine (2010)

Lankun Wu Eric Rowe Ksenija Jeftinija Srdija Jeftinija Ludmila Rizshsky

Abstract Background With a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca 2+ . Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca 2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca 2+ elevation is not influenced by alkamides. Methods A non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca 2+ levels. Changes in cytosolic Ca 2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP 3 ) receptor, were tested to determine the mechanism of this Ca 2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity. Results A rapid transient increase in cytosolic Ca 2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP 3 receptor dependent. Echinacea -evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca 2+ elevation is sustained after the Echinacea -induced Ca 2+ release from intracellular Ca 2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca 2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca 2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity. Conclusions Our data indicate that as yet unidentified constituents from Echinacea stimulate an IP 3 receptor and phospholipase C mediation of cytosolic Ca 2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.

HEK 293 cells

Calcium in biology

Fura-2

Inositol trisphosphate

10.1186/1472-6882-10-72

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Citations (1)

Effects of Leptin on Intracellular Calcium Concentrations in Isolated Porcine Somatotropes

Neuroendocrinology (2004)

Aleksandra Glavaski‐Joksimovic Eric Rowe Ksenija Jeftinija Colin G. Scanes Lloyd L. Anderson

Leptin, the product of the obese gene, is a protein that is secreted primarily from adipocytes. Leptin can influence the function of the pituitary gland through its action on the hypothalamus, but it can also directly act at the level of the pituitary gland. The ability of leptin to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by the application of human growth hormone releasing hormone. Leptin increased [Ca2+]i in porcine somatotropes in a dose-dependent manner. The application of 100 nM leptin for 3 min did not have a significant effect on [Ca2+]i, while a 3-min application of 1 µM leptin increased [Ca2+]i in about 50% of the somatotropes (p < 0.01). The application of a second leptin challenge (1 µM) evoked a response in only 18% of the observed somatotropes. The stimulatory effect of leptin was abolished in low calcium saline and blocked by nifedipine, an L-calcium channel blocker, suggesting an involvement of calcium channels. Pretreatment of the cultures with AG 490, a specific Janus kinase inhibitor, and with SB 203580, a mitogen-activated protein kinase (MAP kinase) inhibitor, abolished the increase in [Ca2+]i evoked by leptin. In the presence of Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, the magnitude of the increase in [Ca2+]i evoked by 1 µM leptin was not significantly changed. However, in the presence of L-NAME only 24% of the somatotropes responded to leptin, while in parallel control cultures 70% of the somatotropes responded to leptin. These results imply an involvement of Janus kinase/signal transducer and activator or transcription, MAP kinase and NOS-signaling pathways in the stimulatory effect of leptin on porcine somatotropes.

Calcium in biology

10.1159/000081843

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Excitatory amino acids are released from rat primary afferent neurons in vitro

Neuroscience Letters (1991)

Srdija Jeftinija Ksenija Jeftinija Fang Liu Stephen R. Skilling David H. Smullin

Capsaicin

Basal (medicine)

10.1016/0304-3940(91)90025-o

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Citations (87)