Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

PNAS 104 (29): 12023-12028

Copyright © 2007 by the National Academy of Sciences.

Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium

Inna Hughes*, Mitsuyoshi Saito{dagger}, Paul H. Schlesinger{dagger}, and David M. Ornitz*,{ddagger}

Departments of *Molecular Biology and Pharmacology and {dagger}Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110


Figure 1
View larger version (29K):
[in this window]
[in a new window]

 
Fig. 1. Overexpression of EGFP-Otop1 in COS7 cells alters the purinergic response. (A) WT COS7 cells (n = 23) show a biphasic response to 200 µM ATP characterized by a sharp peak in [Ca2+]i and a rapid reduction in [Ca2+]i to an elevated plateau (bracket). After removal of purinergic stimulus (wash), [Ca2+]i returns to the prestimulation baseline. Cells transfected with EGFP-Otop1 (n = 17) respond to ATP with an increase in [Ca2+]i to an elevated plateau and a delay in the return of the [Ca2+]i to baseline after removal of ATP (arrow). (B) WT COS7 cells (n = 16) respond to 200 µM UTP with a sharp increase in [Ca2+]i. Cells transfected with EGFP-Otop1 (n = 11) did not respond to UTP. (C) EGFP-Otop1-expressing COS7 cells with an initial fluorescence of <200 EGFP fluorescence units were classified as "low expressers" (blue, n = 23). Cells with EGFP fluorescence units >200 units were grouped as "high expressers" (red, n = 19) and compared with untransfected cells (black, n = 35). (D) Bar plots of the experiment shown in C. Mean and median are indicated by a point and bar, respectively. Range by the box and standard deviation by the error bars [WT (*) versus EGFP-Otop1-low expressers, P < 0.03]. (E) Treatment of the cells in C with 10 µM TG revealed a significant (P < 0.001) difference in IP3 releasable ER Ca2+ stores in low versus high EGFP-Otop1-expressing cells. Despite the low fluorescence/expression for EGFP-Otop1 in the low-expressing cells, the response to 200 µM ATP was identical to that of high-expressing cells. (F) Bar plot of the experiment shown in E. The maximum ratio value (maximum amount of ER Ca2+ released after TG stimulation) differs among WT and EGFP-Otop1 low and high expressers [WT (*) versus EGFP-Otop1 low expressers, P < 0.05]. (G) COS7 cells transfected with both AT1-YFP and FLAG-Otop1 (n = 12) shows a significantly (P < 0.001) reduced P2Y response to 200 µM ATP compared with AT1-YFP transfected cells (n = 20), but retains a response to 20 nM AII. (H) COS7 cells transfected with C5aR (n = 7) respond to 2 µM CC peptide with a rapid and transient increase in [Ca2+]i without disrupting the P2Y-mediated increase in [Ca2+]i in response to 200 µM ATP. Cotransfection of C5aR and EGFP-Otop1 (n = 4) does not alter the 2 µM CC induced increase in [Ca2+]i, but eliminates the P2Y peak in response to 200 µM ATP. The data in A, G, and H are single experiments that were reproduced 3 times. The data in BF are the combined results of three independent experiments.

 

Figure 2
View larger version (37K):
[in this window]
[in a new window]

 
Fig. 2. Unique characteristics of EGFP-Otop1 purinergic-induced increase in [Ca2+]i in COS7 cells. (A) In the absence of extracellular Ca2+ (3 mM EGTA), 200 µM ATP elicits a response in WT cells (n = 10) but not in EGFP-Otop1-expressing cells (n = 7). EGFP-Otop1-expressing COS7 cells in Ca2+-containing media are represented by the dashed line. (B) Two hundred micromolar ATP and ADP elicit similar increases in [Ca2+]i in EGFP-Otop1-expressing cells (n = 14), 200 µM UDP leads to an increase in [Ca2+]i of approximately one-half that seen with ATP or ADP. This is independent of the order of addition of nucleotides. Unlike in EGFP-Otop1-expressing cells, the P2Y component of WT cells (n = 27) remained in a refractory period after the initial treatment with ATP. (C) Treatment of EGFP-Otop1-expressing cells with varying concentrations of ATP (n = 11), ADP (n = 8), UDP (n = 5) leads to different levels of [Ca2+]i 200 sec after addition of agonist. All ratio values are subtracted from baseline values to indicate change in [Ca2+]i in response to stimulus. (D) EGFP-Otop1-expressing COS7 cells respond to 200 µM ATP (n = 11), ADP (n = 8), and UDP (n = 5) with increases in [Ca2+]i, but respond minimally to 200 µM BzATP (n = 18), 200 µM ATP{gamma}S (n = 18), 50 µM {alpha}βMeATP (n = 5), and 50 µM 2-(methylthio) adenosine 5'-triphosphate (2MeSATP) (n = 5). All increases in [Ca2+]i at 200 sec after addition of agonist were normalized to the response to 200 µM ATP. The results of a single experiment are shown and were reproduced at least 2 times.

 

Figure 3
View larger version (37K):
[in this window]
[in a new window]

 
Fig. 3. EGFP-Otop1 activity in ROS and HEK293 cells. (A) UTP (200 µM) and ATP (200 µM) do not elicit increases in [Ca2+]i in untransfected ROS cells (n = 4). Treatment with TG increases [Ca2+]I revealing abundant ER Ca2+ stores. (B) In cells transfected with EGFP-Otop1 (n = 4), 200 µM ATP elicits a slow increase in [Ca2+]i. Removal of stimulus results in an increase in [Ca2+]i before a return to baseline. Subsequent stimulation with ATP leads to an identical response. (C) WT HEK293 cells (n = 11) respond to 400 µM UTP with a brief increase in [Ca2+]i and a rapid return to baseline. Subsequent treatment with 200 µM ATP elicits a similar response. EGFP-Otop1-expressing HEK293 cells (n = 6) do not respond to UTP, and 200 µM ATP elicits a sustained increase in [Ca2+]i that does not return to baseline after removal of stimulus. (D) Treatment of WT (n = 19) and EGFP-Otop1-expressing (n = 16) HEK293 cells with 10 µM TG leads to an increase in [Ca2+]i. WT cells do not respond to 200 µM ATP after TG treatment, whereas EGFP-Otop1 cells show a sustained elevation of [Ca2+]i. Data show the result of single experiments that were reproduced at least 3 times.

 

Figure 4
View larger version (19K):
[in this window]
[in a new window]

 
Fig. 4. Suramin inhibits Otop1 activity in COS7 cells. (A) EGFP-Otop1-expressing cells (n = 17) treated with 100 µM suramin for 20 min have an identical increase in [Ca2+]i in response to 200 µM ATP as WT cells (n = 22). After removal of suramin and ATP (wash), EGFP-Otop1-expressing cells respond to 200 µM ATP similar to that seen in cells never treated with suramin (Fig. 1A). Note that in WT cells the P2Y response to ATP was in the refractory period for the second dose of ATP. (B) COS7 cells expressing EGFP-Otop1 (n = 4) were pretreated with suramin for 10 min and then washed. Subsequent challenge with 200 µM ATP resulted in an increase in [Ca2+]i to an elevated plateau similar to that seen in untreated cells. WT cells (n = 11) are not affected by 100 µM suramin. Data show the result of single experiments that were reproduced at least 3 times.

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882