g Control cells induced by ADP, Dark linesrepresent mean ratio values of Ca2+ responses of cells untreated with P2Y12 antagonist

g Control cells induced by ADP, Dark linesrepresent mean ratio values of Ca2+ responses of cells untreated with P2Y12 antagonist. potentiated by the activity of P2Y12-dependent signaling pathways. This potentiation may be mediated by P2Y12 inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y1 and P2Y12 receptor activity directly depends on the capacitative calcium entrance mechanism. arrowstracerepresents the imply ratio value of the responses of the indicated quantity of cells (Black collection gray collection96-h starved cells, medium graydark grayBlack lineMean ratio value. c Control cells induced by 2MeSADP, Black lineControl cells induced by ADP, gray collection96-h starved cells, cells with strong response,medium graycells with medium response,dark graycells with poor response). Mean ratio value. g Control cells induced by ADP, Black linesrepresent mean ratio values of Ca2+ responses of cells untreated with P2Y12 antagonist. a, c, e Nonstarved cells, respectively, Gray linesin the same panels represent mean ratio value of Ca2+ responses of cells pre-treated for Antitumor agent-3 3?min with 10?M AR-C69931MX (a, ?=? 95; b, arrowsblack linegray linetracerepresents mean ratio value from five experiments. ***minus signnnnnnminus signColour of barsas explained above. Difference between bars marked with aminus signis statistically insignificant To distinguish if the P2Y12 receptor plays an active role in the calcium signal formation or if it only modulates the result of P2Y1 receptor activity, we used AR-C69931MX in two individual experimental setups. In the first experiment, P2Y12 receptor competitive antagonist was used before addition of agonist to inhibit both hypothetical P2Y12 direct calcium signalling as well as regulation of P2Y1 activity by P2Y12 receptor. In the second experiment, the use of antagonist well after agonist addition but before medium replacement with that containing calcium should affect only regulatory functions of P2Y12 receptor but not its ability to directly form the calcium signal. As has been shown, AR-C69931MX has an inhibitory effect on the second phase of Ca2+ response, however this effect does not depend on the moment of the antagonist addition (cells treated with AR-C69931MX before ADP addition: 1.43??0.44?AU, em n /em ?=?12; cells treated with Antitumor agent-3 AR-C69931MX after ADP addition: 1.55??0.42?AU, em n /em ?=?18; the difference was not statistically significant) (Fig.?5b). Physique?5b also shows that AR-C69931MX has no statistically significant effect on the first phase of the calcium response. The same results were observed when 2MeSADP was used as an agonist (data not shown). Conversation It is now well-documented that in the Gq-dependent signalling initiated by ADP or 2MeSADP, the P2Y1 receptor activation triggers PLC activation and increase [9C17]. On the other hand, the same agonists, via the P2Y12 receptor, activate the Gi pathway and inhibit adenylate cyclase in various animal cells [11, 12, 18, 24, 25]. The cross-talk between those two receptors is extremely complex [9, 26]. In human platelets, Sage et al. [27] and Fox et al. [28] suggested that P2Y12 may enhance P2Y1-induced cytosolic Ca2+ rise, whereas Daniel et al. [29] offered evidence that this receptor is not involved in such response. Hardy et al. [30] have explained this conflicting evidence as the different conditions used during platelets preparation. Similarly in glioma C6 cells, there is conflicting evidence regarding the role of P2Y1 in ADP-mediated calcium response that can also be explained by the differences in the culture conditions [10]. Presence or absence of serum in the culture medium provides conclusions on functional activity [9C11] or inactivity [18] of this receptor. Hardy et al. [30], as well as Sage et al. [27], suggested the modulatory role of P2Y12, positively regulating P2Y1-induced Ca2+ response. It has Rabbit polyclonal to CLOCK been suggested that this potentiation is usually mediated by P2Y12-induced inhibition of adenylate cyclase and activation of phosphatidylinositol 3-kinase (PI3-K), whereas the effect of P2Y1 on PI3-K is usually inhibitory [30]. Our previous study concerning cross-talk between nucleotide receptor-induced signalling pathways in glioma C6 cells also revealed P2Y1 inhibitory and P2Y12 stimulatory effects on PI3-K signalling [9, 10]. Thus, since activation of the P2Y12 receptor in glioma C6 inhibits adenylate cyclase [11, 12] and.***minus signnnnnnminus signColour of barsas explained above. mean ratio value of the responses of the indicated quantity of cells (Black collection gray collection96-h starved cells, medium graydark grayBlack lineMean ratio value. c Control cells induced by 2MeSADP, Black lineControl cells induced by ADP, gray collection96-h starved cells, cells with strong response,medium graycells with medium response,dark graycells with poor response). Mean ratio value. g Control cells induced by ADP, Black linesrepresent mean ratio values of Ca2+ responses of cells untreated with P2Y12 antagonist. a, c, e Nonstarved cells, respectively, Gray linesin the same panels represent mean ratio value of Ca2+ responses of cells pre-treated for 3?min with 10?M AR-C69931MX (a, ?=? 95; b, arrowsblack linegray linetracerepresents mean ratio value from five experiments. ***minus signnnnnnminus signColour of barsas explained above. Difference between bars marked with aminus signis statistically insignificant To distinguish if the P2Y12 receptor plays an active role in the calcium signal formation or if it only modulates the result of P2Y1 receptor activity, we used AR-C69931MX in two individual experimental setups. In the first experiment, P2Y12 receptor competitive antagonist was used before addition of agonist to inhibit both hypothetical P2Y12 direct calcium signalling as well as regulation of P2Y1 activity by P2Y12 receptor. In the second experiment, the use of antagonist well after agonist addition but before medium replacement with that containing calcium should affect only regulatory functions of P2Y12 receptor but not its ability to directly form the calcium signal. As has been shown, AR-C69931MX has an inhibitory effect on the second phase of Ca2+ response, however this effect does not depend on the moment of the antagonist addition (cells treated with AR-C69931MX before ADP addition: 1.43??0.44?AU, em n /em ?=?12; cells treated with AR-C69931MX after ADP addition: 1.55??0.42?AU, em n /em ?=?18; the difference was not statistically significant) (Fig.?5b). Physique?5b also shows that AR-C69931MX has no statistically significant effect on the first phase of the calcium response. The same results were observed when 2MeSADP was used Antitumor agent-3 as an agonist (data not shown). Discussion It is now well-documented that in the Gq-dependent signalling initiated by ADP or 2MeSADP, the P2Y1 receptor activation triggers PLC activation and increase [9C17]. On the other hand, the same agonists, via the P2Y12 receptor, activate the Gi pathway and inhibit adenylate cyclase in various animal cells [11, 12, 18, 24, 25]. The cross-talk between those two receptors is extremely complex [9, 26]. In human platelets, Sage et al. [27] and Fox et al. [28] suggested that P2Y12 may enhance P2Y1-induced cytosolic Ca2+ rise, whereas Daniel et al. [29] offered evidence that this receptor is not involved in such response. Hardy et al. [30] have explained this conflicting evidence as the different conditions used during platelets preparation. Similarly in glioma C6 cells, there is conflicting evidence regarding the role of P2Y1 in ADP-mediated calcium response that can also be explained by the differences in the culture conditions [10]. Presence or absence of serum in the culture medium provides conclusions on functional activity [9C11] or inactivity [18] of this receptor. Hardy et al. [30], as well as Sage et al. [27], suggested the modulatory role of P2Y12, positively regulating P2Y1-induced Ca2+ response. It has been suggested that this potentiation is mediated by P2Y12-induced inhibition of adenylate cyclase and activation of phosphatidylinositol 3-kinase (PI3-K), whereas the effect of P2Y1 on PI3-K is inhibitory [30]. Our previous study concerning cross-talk between nucleotide receptor-induced signalling pathways in glioma C6 cells also revealed P2Y1 inhibitory and P2Y12 stimulatory effects on PI3-K signalling [9, 10]. Thus, since stimulation of the P2Y12 receptor in glioma C6 inhibits adenylate cyclase [11, 12] and stimulates PI3-K [9, 10], its modulatory effect on the P2Y1-induced Ca2+ responses in this cell line may occur via a similar mechanism to the one suggested in platelets [30]. It has been proposed that in this process the cAMP-dependent pathway has a stimulatory effect on PM calcium pumps, thereby limiting the strength of the calcium response. The P2Y12 receptor reduced this effect by inhibition of adenylate cyclase activity. Hardy et al. [30] suggested that PI3-K, activated by the P2Y12 receptor stimulation, may also activate PLC, leading to the rise in PIP3 and enhancement of the calcium signal. It has been reported that receptors coupled to PI3-K.