1Departments of Neurosurgery and 2Pharmacology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan
Submitted 25 August 2003 ; accepted in final form 29 October 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
norepinephrine; 1A-adrenergic receptor; nonselective cation channel; G13 protein; arachidonic acid release
![]() |
MATERIAL AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurement of [Ca2+]i. [Ca2+]i was measured using a fluorescent probe fluo 3. The measurements of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan) and an Attofluor Ratio-Vision real time digital fluorescence analyzer (Atto Instruments, Potomac, MD) were performed exactly as described previously (7).
Microinjection of G12G228A and G13G225A. G12G228A and G13G225A in pcDNA 3.1(+) were constructed as described previously (8, 14). Microinjection of G12G228A and G13G225A was performed as described previously (8, 14). Briefly, cells were seeded onto glass coverslips coated with fibronectin (Iwaki Glass, Chiba, Japan), which were marked with a cross to facilitate the localization of injected cells, and incubated overnight in Ham's F-12 medium containing 1% FCS. Plasmids (100 ng/µl) encoding for G12G228A and G13G225A were microinjected into cell nuclei. As a control, expression plasmids without inserts were microinjected in an adjacent field on the same coverslip. Microinjection was performed using a manual microinjection system (Eppendorf-5 Prime, Hamburg, Germany) equipped with an Axiovert 100 inverted microscope (Carl-Zeiss, Frankfurt, Germany).
Transfection of G12G228A and G13G225A. For transient expression of G12G228A or G13G225A, cells were transfected with plasmid (100 ng/µl) encoding for G12G228A or G13G225A by MBS Mammalian Transfection Kit (Stratagene, CA) according to the manufacturer's instructions. After 24 h incubation, we used these cells for measurement of [3H]arachidonic acid release.
[3H]arachidonic acid release. The level of [3H]arachidonic acid release was determined as described previously (12). Briefly, cells in 100-mm dishes were incubated overnight with [3H]arachidonic acid (final concentration, 1 µCi/ml). After washing, norepinephrine was added for 5 min. The medium was then removed, acidified with 100 µl of 1N formic acid, and extracted with 3 ml of chloroform. The extracts were evaporated to dryness, resuspended in 50 µl of chloroform, and applied to silica gel plates for thin-layer chromatography (Merck, Darmstadt, Germany). The plates were developed in heptane/diethyl ether/acetic acid (vol/vol; 75:25:4). The distance of movement was visualized with iodine vapor. The plate was scraped, and the radioactivity was counted with a liquid scintillation counter.
Materials. Constitutively active G12 or G13 in pcDNA3(+) was kindly provided by Dr. Manabu Negishi (Kyoto University, Japan). Welfide (Osaka, Japan) kindly provided Y-27632. Other chemicals were obtained commercially.
Statistical analysis. All results were expressed as means ± SE. The data were subjected to a two-way analysis of variance. When a significant F value was encountered, the Newman-Keuls' multiple range test was used to test for significant differences between treatment groups. A probability level of P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Effects of G12G228A and G13G225A on norepinephrine-induced activation of Ca2+ channels. To investigate whether G12 and G13 are involved in the activation of NSCCs, we investigated the effects of G12G228A and G13G225A on the norepinephrine-induced increase in [Ca2+]i in CHO-1A. In this experiment, G12G228A and G13G225A were microinjected into CHO-
1A, and the norepinephrine-induced increase in [Ca2+]i in these cells was analyzed using Attofluor Ratio-Vision real time digital fluorescence analyzer. Norepinephrine at 100 nM induced a biphasic increase in [Ca2+]i consisting of an initial transient peak and a subsequent sustained increase in CHO-
1A microinjected with G12G228A (Fig. 2A). On the other hand, 100 nM norepinephrine induced only a transient increase in [Ca2+]i in CHO-
1A microinjected with G13G225A (Fig. 2, B and D). The magnitudes of transient increase in [Ca2+]i in CHO-
1A microinjected with G13G225A were similar to those in CHO-
1A and CHO-
1A microinjected with G12G228A (Fig. 2C). On the other hand, norepinephrine failed to induce a sustained increase in [Ca2+]i in CHO-
1A microinjected with constitutively active G12 or G13 (data not shown).
|
Effects of Y-27632 and wortmannin on norepinephrine-induced activation of Ca2+ channels. According to the data using G13G225A, G13 plays important roles in NSCCs activation by norepinephrine. It is generally accepted that Rho/Rho-kinase (ROCK) pathway is a downstream target of G13 (21). We examined the effects of ROCK on norepinephrine-induced increase in [Ca2+]i in CHO-1A. In this experiment, Y-27632 was used as a specific inhibitor of ROCK (22). Y-27632 at 10 µM did not affect to the norepinephrine-induced transient and sustained increase in [Ca2+]i (Fig. 3, A-C).
|
Based on the previous data that phosphoinoditide 3-kinase (PI3K) is involved in some types of NSCCs activation by endothelin-1 in CHO cells stably expressing endothelin receptors (9, 10), we examined the effects of wortmannin, an inhibitor of PI3K, on norepinephrine-induced increase in [Ca2+]i in CHO-1A. Wortmannin at 1 µM failed to inhibit the norepinephrine-induced transient and sustained increase in [Ca2+]i (Fig. 3, D-F).
Effects of G12G228A and G13G225A on norepinephrine-induced arachidonic acid release. G12G228A or G13G225A was transiently transfected for evaluating the role of G12 or G13, respectively, in norepinephrine-induced arachidonic acid release. For this purpose, we used MBS Mammalian Transfection Kit. When we transfected green fluorescent protein (GFP) with this method, around 65% of cells were GFP positive (data not shown). The magnitudes of norepinephrine-induced arachidonic acid release in CHO-1A transfected with G13G225A were around 20% of those in CHO-
1A (Fig. 4). In contrast, G12G228A failed to inhibit norepinephrine-induced arachidonic acid release (Fig. 4). The magnitudes of norepinephrine-induced arachidonic acid release in CHO-
1A transfected with only vector were similar to those in CHO-
1A (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extracellular Ca2+ influx through NSCCs plays a critical role for norepinephrine-induced arachidonic acid release in CHO-1A (12). Therefore, we examined the effects of G13 on norepinephrine-induced arachidonic acid release. Disruption of signaling through endogenous G13 by its dominant negative mutant (G13G225A) inhibited norepinephrine-induced arachidonic acid release in CHO-
1A (Fig. 4), indicating that activation of arachidonic acid release is mediated by G13. In contrast, G12G228A failed to inhibit norepinephrine-induced arachidonic acid release (Fig. 4). Therefore, G13, but not G12, plays important roles in norepinephrine-induced arachidonic acid release. Norepinephrine-induced arachidonic acid release was not inhibited completely by G13G225A in this study (Fig. 4). We think that this is because G13G225A is not transfected to all cells. However, another possibility is that norepinephrine induces arachidonic acid release with another unknown pathway in CHO-
1A. Further research is necessary to confirm this.
Next, we summarized the pharmacological characteristics and activation mechanisms of Ca2+ channels activated by norepinephrine in CHO-1A and by endothelin-1 in CHO-ETA and CHO-ETB (Table 1). On the basis of the sensitivity to Ca2+ channel blockers, SK&F 96365 and LOE 908, NSCCs activated by norepinephrine in CHO-
1A (11) and NSCC-1 activated by endothelin-1 in CHO-ETA and CHO-ETB (15) have the same pharmacological sensitivities (LOE 908-sensitivity and SK&F 96365-resistance). In addition, the activation mechanisms of NSCCs by norepinephrine in CHO-
1A (Fig. 2) are similar to those of NSCC-1 in CHO-ETB (8) at the following points. 1) Both channels are activated via G13-dependent and Gq/PLC-independent pathways. 2) Neither Rocknor PI3K-dependent pathway are involved in these channels' activation. These results indicate that
1A-ARs and endothelinB receptors may activate some types of NSCCs (NSCC-1) via the same pathways in CHO cells. Moreover, NSCCs activated by norepinephrine are included in NSCC-1.
|
![]() |
ACKNOWLEDGMENTS |
---|
GRANTS
This study was supported by a grant from the Smoking Research Foundation, Japan, and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Berridge MJ. Inositol triphosphate and calcium signalling. Nature 361: 315-325, 1993.[CrossRef][ISI][Medline]
3. Burch RM, Luini A, Mais DE, Corda D, Vanderhoek JY, Kohn LD, and Axelrod J. Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2. J Biol Chem 261: 11236-11241, 1986.
4. Davis JN, Arnett CD, Hoyler E, Stalvey LP, Daly JW, and Skolnick P. Brain alpha-adrenergic receptors: comparison of [3H]WB 4101 binding with noradrenaline-stimulated cyclic AMP accumulation in rat cerebral cortex. Brain Res 159: 125-135, 1978.[CrossRef][ISI][Medline]
5. Encabo A, Romanin C, Birke FW, Kukovetz WR, and Groschner K. Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908. Br J Pharmacol 119: 702-706, 1996.[Abstract]
6. Gardner P. Calcium and T lymphocyte activation. Cell 59: 15-20, 1989.[ISI][Medline]
7. Kawanabe Y, Hashimoto N, and Masaki T. Ca2+ channels involved in endothelin-induced mitogenic response in carotid artery vascular smooth muscle cells. Am J Physiol Cell Physiol 282: C330-C337, 2002.
8. Kawanabe Y, Hashimoto N, and Masaki T. Characterization of G proteins involved in activation of nonselective cation channels by endothelinB receptor. Br J Pharmacol 136: 1015-1022, 2002.
9. Kawanabe Y, Hashimoto N, and Masaki T. Effects of phosphoinositide 3-kinase on the endothelin-1-induced activation of voltage-independent Ca2+ channels and mitogenesis in Chinese hamster ovary cells stably expressing endothelinA receptor. Mol Pharmacol 62: 756-761, 2002.
10. Kawanabe Y, Hashimoto N, and Masaki T. Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor. Am J Physiol Cell Physiol 284: C506-C510, 2003.
11. Kawanabe Y, Hashimoto N, and Masaki T. Molecular mechanisms for activation of Ca2+-permeable nonselective cation channels by endothelin-1 in C6 glioma cells. Biochem Pharmacol 65: 1435-1439, 2003.[CrossRef][ISI][Medline]
12. Kawanabe Y, Hashimoto N, Masaki T, and Miwa S. Ca2+ influx through nonselective cation channels plays an essential role in noradrenaline-induced arachidonic acid release in Chinese hamster ovary cells expressing 1A-,
1B-, or
1D-adrenergic receptors. J Pharmacol Exp Ther 299: 901-907, 2001.
13. Kawanabe Y, Hashimoto N, Miwa S, and Masaki T. Effects of Ca2+ influx through nonselective cation channel on noradrenaline-induced mitogenic responses. Eur J Pharmacol 447: 31-36, 2002.[CrossRef][ISI][Medline]
14. Kawanabe Y, Nozaki K, Hashimoto N, and Masaki T. Characterization of Ca2+ channels and G proteins involved in arachidonic acid release by endothelin-1/endothelinA receptor. Mol Pharmacol 64: 689-695, 2003.
15. Kawanabe Y, Okamoto Y, Enoki T, Hashimoto N, and Masaki T. Ca2+ channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors. Am J Physiol Cell Physiol 281: C1676-C1685, 2001.
16. Kawanabe Y, Okamoto Y, Miwa S, Hashimoto N, and Masaki T. Molecular mechanisms for the activation of voltage-independent Ca2+ channels by endothelin-1 in Chinese hamster ovary cells stably expressing human endothelinA receptors. Mol Pharmacol 62: 75-80, 2002.
17. Kawanabe Y, Okamoto Y, Nozaki K, Hashimoto N, Miwa S, and Masaki T. Molecular mechanism for endothelin-1-induced stress-fiber formation: analysis of G proteins using a mutant endothelinA receptor. Mol Pharmacol 61: 277-284, 2002.
18. Llahi S and Fain JN. Alpha1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex. J Biol Chem 267: 3679-3685, 1992.
19. Maruyama Y, Nishida M, Sugimoto Y, Tanabe S, Turner JH, Kozasa T, Wada T, Nagao T, and Kurose H. Galpha12/13 mediates alpha1-adrenergic receptor-induced cardiac hypertrophy. Circ Res 91: 961-969, 2002.
20. Meritt JE, Airmstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, Mccarthy SA, Moores KE, and Rink TJ. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271: 515-522, 1990.[ISI][Medline]
21. Seasholtz TM, Majumdar M, and Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 55: 949-956, 1999.
22. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishima T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997.[CrossRef][ISI][Medline]
23. Wilson KM and Minneman KP. Different pathways of [3H]inositol phosphate formation mediated by alpha1a- and alpha1b-adrenergic receptors. J Biol Chem 265: 17601-17606, 1990.
24. Wu D, Katz A, Lee CH, and Simon MI. Activation of phospholipase C by alpha1-adrenergic receptors is mediated by the alpha subunits of Gq family. J Biol Chem 267: 25798-25802, 1992.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |