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[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.

Sun XC, Cui M, Bonanno JA - BMC Physiol. (2004)

Bottom Line: Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively.HCO3- not only directly activates sAC, but also up-regulates the expression of sAC.These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu

ABSTRACT

Background: Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.

Results: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).

Conclusion: HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

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The effect of HCO3--induced sAC expression on apical Cl- permeability in BCECs A: HCO3--starved BCECs grown on permeable Anodisc. Both apical (AP) and basolateral (BL) compartments were initially perfused with Cl-- and HCO3--free Ringer solution. After the 1st apical (AP) Cl- pulse, HCO3--rich Ringer solution (BR) was introduced on both sides for at least 5 min before the 2nd Cl- pulse. Break in trace indicates period of wash in Cl--free and BR solution until trace stabilized (at least 5 min). B: maximum slope summary data for A (n = 5); all fluorescence values were normalized to the fluorescence value in the absence of Cl- (F0) obtained just before addition of Cl-. Calculated slopes were adjusted by any background drift in the fluorescence trace that was apparent just before addition of Cl-. C: BCECs cultured in BR medium, same experiment as in A. D: summary data for C (n = 5). *Significantly different from HCO3--free solution (BF) (P < 0.05).
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Figure 6: The effect of HCO3--induced sAC expression on apical Cl- permeability in BCECs A: HCO3--starved BCECs grown on permeable Anodisc. Both apical (AP) and basolateral (BL) compartments were initially perfused with Cl-- and HCO3--free Ringer solution. After the 1st apical (AP) Cl- pulse, HCO3--rich Ringer solution (BR) was introduced on both sides for at least 5 min before the 2nd Cl- pulse. Break in trace indicates period of wash in Cl--free and BR solution until trace stabilized (at least 5 min). B: maximum slope summary data for A (n = 5); all fluorescence values were normalized to the fluorescence value in the absence of Cl- (F0) obtained just before addition of Cl-. Calculated slopes were adjusted by any background drift in the fluorescence trace that was apparent just before addition of Cl-. C: BCECs cultured in BR medium, same experiment as in A. D: summary data for C (n = 5). *Significantly different from HCO3--free solution (BF) (P < 0.05).

Mentions: Acute exposure to HCO3- activates sAC, increases [cAMP] and PKA activity, leading to the increased phosphorylation of CFTR and increased apical Cl- permeability [18]. Since culturing in the absence of HCO3- can down-regulate sAC expression and intracellular [cAMP] in BCECs, acute exposure of bicarbonate-starved cells to HCO3- should show a reduced increase in apical Cl- permeability relative to cells cultured in the presence of bicarbonate. To test this possibility, we loaded bicarbonate-starved BCECs with the halide-sensitive dye MEQ and measured the rate of fluorescence change in response to apical Cl- pulses. Both apical and basolateral sides were initially perfused with Cl- and HCO3--free solutions. As shown in figure 6A, when Cl- was added to the apical side for 90s, a small, slow decrease in MEQ fluorescence, caused by the entry of Cl-, was observed. Both sides were then bathed in Cl--free, HCO3--rich solution for at least 5 min. When the fluorescence signal had stabilized, Cl- was applied to the apical side in the presence of HCO3-. The decrease in MEQ fluorescence was not significantly changed relative to the paired control. Figure 6B summarizes the results and shows that the apical Cl- permeability of HCO3--starved BCEC was increased by 10.3% in the presence of HCO3-. However, the apical Cl- permeability in BCECs cultured in bicarbonate, as shown in figure 6C, was significantly increased relative to the paired control. Figure 6D summarizes the results and shows that the apical Cl- permeability of BCECs cultured in bicarbonate was increased by 78% in the presence of HCO3-.


[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.

Sun XC, Cui M, Bonanno JA - BMC Physiol. (2004)

The effect of HCO3--induced sAC expression on apical Cl- permeability in BCECs A: HCO3--starved BCECs grown on permeable Anodisc. Both apical (AP) and basolateral (BL) compartments were initially perfused with Cl-- and HCO3--free Ringer solution. After the 1st apical (AP) Cl- pulse, HCO3--rich Ringer solution (BR) was introduced on both sides for at least 5 min before the 2nd Cl- pulse. Break in trace indicates period of wash in Cl--free and BR solution until trace stabilized (at least 5 min). B: maximum slope summary data for A (n = 5); all fluorescence values were normalized to the fluorescence value in the absence of Cl- (F0) obtained just before addition of Cl-. Calculated slopes were adjusted by any background drift in the fluorescence trace that was apparent just before addition of Cl-. C: BCECs cultured in BR medium, same experiment as in A. D: summary data for C (n = 5). *Significantly different from HCO3--free solution (BF) (P < 0.05).
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Figure 6: The effect of HCO3--induced sAC expression on apical Cl- permeability in BCECs A: HCO3--starved BCECs grown on permeable Anodisc. Both apical (AP) and basolateral (BL) compartments were initially perfused with Cl-- and HCO3--free Ringer solution. After the 1st apical (AP) Cl- pulse, HCO3--rich Ringer solution (BR) was introduced on both sides for at least 5 min before the 2nd Cl- pulse. Break in trace indicates period of wash in Cl--free and BR solution until trace stabilized (at least 5 min). B: maximum slope summary data for A (n = 5); all fluorescence values were normalized to the fluorescence value in the absence of Cl- (F0) obtained just before addition of Cl-. Calculated slopes were adjusted by any background drift in the fluorescence trace that was apparent just before addition of Cl-. C: BCECs cultured in BR medium, same experiment as in A. D: summary data for C (n = 5). *Significantly different from HCO3--free solution (BF) (P < 0.05).
Mentions: Acute exposure to HCO3- activates sAC, increases [cAMP] and PKA activity, leading to the increased phosphorylation of CFTR and increased apical Cl- permeability [18]. Since culturing in the absence of HCO3- can down-regulate sAC expression and intracellular [cAMP] in BCECs, acute exposure of bicarbonate-starved cells to HCO3- should show a reduced increase in apical Cl- permeability relative to cells cultured in the presence of bicarbonate. To test this possibility, we loaded bicarbonate-starved BCECs with the halide-sensitive dye MEQ and measured the rate of fluorescence change in response to apical Cl- pulses. Both apical and basolateral sides were initially perfused with Cl- and HCO3--free solutions. As shown in figure 6A, when Cl- was added to the apical side for 90s, a small, slow decrease in MEQ fluorescence, caused by the entry of Cl-, was observed. Both sides were then bathed in Cl--free, HCO3--rich solution for at least 5 min. When the fluorescence signal had stabilized, Cl- was applied to the apical side in the presence of HCO3-. The decrease in MEQ fluorescence was not significantly changed relative to the paired control. Figure 6B summarizes the results and shows that the apical Cl- permeability of HCO3--starved BCEC was increased by 10.3% in the presence of HCO3-. However, the apical Cl- permeability in BCECs cultured in bicarbonate, as shown in figure 6C, was significantly increased relative to the paired control. Figure 6D summarizes the results and shows that the apical Cl- permeability of BCECs cultured in bicarbonate was increased by 78% in the presence of HCO3-.

Bottom Line: Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively.HCO3- not only directly activates sAC, but also up-regulates the expression of sAC.These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu

ABSTRACT

Background: Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.

Results: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).

Conclusion: HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

Show MeSH
Related in: MedlinePlus