Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets

Abstract

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

Dynamics of Ca 2+ i and pH i in Ehrlich ascites tumor cells after Ca 2+ -mobilizing agonists or exposure to hypertonic solution

“…Moreover, we have previously shown that the effects of thrombin and bradykinin on pH i in Ehrlich cells are only marginal at 25°C [26]. The present study demonstrates that at 37°C, these agonists elicited a biphasic change in pH i , similar to that seen in a number of other cell types studied [15,29,45]. The initial acidification is likely to involve a hyperpolarization-induced influx of H + , as well as an increase in metabolic H + production, both of which are expected to result from the agonist-induced increase in [Ca 2+ ] i .…”

“…Studies in other cell types have produced conflicting evidence regarding the qualitative as well as the temporal relationship between changes in pH i and [Ca 2+ ] i induced by these agonists. Thus, inhibition of the Ca 2+ transient using chelators of intracellular Ca 2+ has been reported to result in no [41], partial [15], or complete [29] inhibition of the thrombin-induced pH i response. Conversely, the increase in [Ca 2+ ] i in response to thrombin has been suggested to be either secondary to activation of the Na + /H + exchanger and subsequent intracellular alkalinization [36], or independent of the alkalinization [1].…”

“…In many cells, the Na + /H + exchanger is activated after exposure to these agonists, resulting in intracellular alkalinization [23,29,45]. Studies in other cell types have produced conflicting evidence regarding the qualitative as well as the temporal relationship between changes in pH i and [Ca 2+ ] i induced by these agonists.…”

Interdependence of Ca2+ and proton movements in trout hepatocytes

“…Accordingly, the mobilization of Ca 2+ modified the proton distribution across the cell membrane. This observation is not in line with previous studies on other cells in which an alkalinization induced by Ca 2+ mobilizing was reported to be due to the activation of NHE (Poch et al, 1993;Martin-Requero et al, 1997) or Ca 2+ /H + exchange (Schulz et al, 1989;Anwer, 1993;Daugirdas et al, 1995;Ouyang et al, 1995;Yamada et al, 1996;Alfonso et al, 2005) ] i did not prevent, but attenuated, the thapsigargin-induced alkalinization, while in the presence of ionomycin the alkalinization was almost abolished. This indicates that the response to thapsigargin is not completely due to Ca 2+ changes, and contributions from other mechanisms cannot be excluded.…”

“…A number of studies investigating the possible linkage between changes in [Ca 2+ ] i and pHi revealed that the nature of this linkage appears to be dependent on the cell type, as shown for example by analyzing the effect of changes in [Ca 2+ ] i on pHi. While an alkalinization induced by Ca 2+ mobilizing agents has been reported to be dependent on extracellular Ca 2+ (Ca 2+ e ) in rat lymphocytes , chicken granulose cells (Asem et al, 1992), rat hepatocytes (Anwer, 1993), cortical neurons (Ouyang et al, 1995), rabbit renal proximal tubules (Yamada et al, 1996) and human platelets (Poch et al, 1993), the absence of Ca 2+ e did not prevent that alkalinization in human lymphocytes (Cabado et al, 2000).…”