J Korean Ophthalmol Soc.  2007 Aug;48(8):1134-1142.

Identification of P2Y11 Receptor Expressed in Human Retinoblastoma Cells

• Kim YH
• Kim DR
• Chun KI
• Lee JH
• Kong ID

Abstract

PURPOSE: The present study aimed to identify the characteristics and physiological function of the P2Y11 receptor, a receptor likely expressed in human retinoblastoma cells.
METHODS
We measured possible P2Y11 signaling in WERI-Rb-1 cells using a Ca2+ imaging technique and RT-PCR.
RESULTS
1) 10 micro M ATP elicited a strong but transient increase in Ca2+ in the WERI-Rb-1 cells, and this Ca2+ rise was well maintained after external Ca2+-depletion. 2) ATP-induced Ca2+ response arose entirely through Ca2+ mobilization. 3) P2Y11 agonist (BzATP, 100 micro M) increased Ca2+ by 31.2+/-3.7 % of ATP effect. 4) mRNA for P2Y11 subtype was identified using RT-PCR.
CONCLUSIONS
P2Y11 purinergic activation can increase the intracellular calcium level through calcium mobilization in undifferentiated retinoblastoma cells, which may play an important role in cell proliferation, differentiation, and even pathologic processes.

Keyword

Calcium; P2Y11 receptor; Retinoblastoma cell

MeSH Terms

Adenosine Triphosphate
Calcium
Cell Proliferation
Humans*
Pathologic Processes
Retinoblastoma*
RNA, Messenger
Adenosine Triphosphate
Calcium
RNA, Messenger

Figure

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  Figure 1. ATP-induced [Ca2+] response in WERI-Rb-1 cells. WERI-Rb-1 cells, which was loaded with 5 pM Fluo-3/AM for lhour, was mounted in a flow chamber. A: 100 pM ATP increased intracellular calcium level (F/F0=7.1±0.2), which response was not desensitized by additional ATP application. There were not significant differences in the peak amplitude of [Ca2+]i responses among 3 repeated ATP application. F0=basal fluorescence value loaded by Flu-3/AM, F=fluorescence changed after introducing ATP, n=total cell number, N=experimental number, solid line=the duration for applying ATP.

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  Figure 2. Effect of Thapsigargin (TG) on ATP-induced [Ca2+]i increase in WERI-Rb-1 cells. Original trace (A) and histogram (B) showing an almost complete suppression of maximal amplitude of 10 pM ATP-induced calcium transient after pretreatment with 1 pM thapsigargin (TG). Drugs were applied for 9 min as indicated by thick bars. F0=basal fluorescence value loaded by Flu-3/AM, F=fluorescence changed after introducing ATP, n=total cell number, N=experimental number, solid line=the duration for applying TG.jkos-48-1134* .001)

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  Figure 3. Differential effect of P2Y and P2X purinoceptor agonists on [Ca2+]i in WERI-Rb-1 cells. Original trace (A) and histogram (B) showing 2MeS-ATP (1 pM), P2Y agonist, induced calcium transient, but those intracellular calcium rise was not represented by the application of the P2X agonist, ap-MeATP (100 pM). F0=basal fluorescence value loaded by Flu-3/AM, F=fluorescence changed after introducing agonists, n=total cell number, N=experimental number, solid line=the duration for applying agonists.

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  Figure 4. Effect of BzATP on [Ca2+]i increase in WERI-Rb-1 cells. Original trace (A) and histogram (B) showing BzATP-induced [Ca2+]i rise was almost to 31.2±3.7% of maximal amplitude of 10 pM ATP -induced calcium transient, and BzATP-induced [Ca]i response was similar in the incubation of calcium free solution (0 Ca2+). F0=basal fluorescence value loaded by Flu-3/AM, F=fluorescence changed after introducing agonists, n=total cell number, N=experimental number, solid line=the duration for applying agonists.

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  Figure 5. RT-PCR analysis of mRNA encoding the subunits of P2Y receptors expressed in WERI-Rb-1 cells. Total RNA isolated from WERI-Rb-1 cells were reverse transcribed and amplified by PCR with specific primers to the subunits of P2Y receptors. The resultant PCR products were visualized on agarose gel, containing ethidium bromide. As an internal control, GAPDH was also amplified. Estimated product size for each subunit is shown in parenthesis. M, DNA size marker.

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