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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 effects of bath pH and intracellular [cAMP] on sAC expression. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different extracellular pH. From left to right: M: Marker; pH: 7.0, 7.5 and 8.0. B. immunoblots of sAC protein expression and β-actin from Calu-3 cells exposed to HCO3--free culture medium at pH 7.0, 7.5 or 8.0. C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to 10 μM adenosine or 10 μM forskolin in the absence of bicarbonate. From left to right: M: Marker; SBF: standard bicarbonate free media; Ado: adenosine; FSK: forskolin. D: summary of the ratios of band densities (sAC/GAPDH) from A and C. Error bar indicates ± SE (n = 3).
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Figure 3: The effects of bath pH and intracellular [cAMP] on sAC expression. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different extracellular pH. From left to right: M: Marker; pH: 7.0, 7.5 and 8.0. B. immunoblots of sAC protein expression and β-actin from Calu-3 cells exposed to HCO3--free culture medium at pH 7.0, 7.5 or 8.0. C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to 10 μM adenosine or 10 μM forskolin in the absence of bicarbonate. From left to right: M: Marker; SBF: standard bicarbonate free media; Ado: adenosine; FSK: forskolin. D: summary of the ratios of band densities (sAC/GAPDH) from A and C. Error bar indicates ± SE (n = 3).

Mentions: Changes in extracellular pH can regulate gene expression in renal cells in vitro [22]. Since culture medium pH varies with different [HCO3-], the up-regulation of sAC expression could be induced by changes in intracellular pH (pHi) secondary to differences in bath pH rather than HCO3-. The bath pH at different [HCO3-] was determined empirically. The bath pH ranges from 6.8 at 0 HCO3- to 7.8 at 80 HCO3-, if the culture medium contains 25 mM Hepes. A full pH unit difference in bath pH causes ~0.45 pH unit change in pHi [18]. In figure 3A, bath pH ranging from 7.0 to 8.0 did not change the expression of sAC mRNA in BCECs cultured in the absence of HCO3-. The ratios of competitive PCR product band densities (sAC/GAPDH) are summarized in Figure 3D. To examine sAC protein expression in different bath pH, sAC immunoblots were employed using Calu-3 cells. The effect of varying culture medium pH on sAC protein expression in Calu-3 cells is shown in figure 3B. From pH 7.0 to 8.0, equal density of sAC immunoblot bands were observed, which is consistent with the results shown in Fig. 3A.


[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 effects of bath pH and intracellular [cAMP] on sAC expression. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different extracellular pH. From left to right: M: Marker; pH: 7.0, 7.5 and 8.0. B. immunoblots of sAC protein expression and β-actin from Calu-3 cells exposed to HCO3--free culture medium at pH 7.0, 7.5 or 8.0. C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to 10 μM adenosine or 10 μM forskolin in the absence of bicarbonate. From left to right: M: Marker; SBF: standard bicarbonate free media; Ado: adenosine; FSK: forskolin. D: summary of the ratios of band densities (sAC/GAPDH) from A and C. Error bar indicates ± SE (n = 3).
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Related In: Results  -  Collection

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Figure 3: The effects of bath pH and intracellular [cAMP] on sAC expression. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different extracellular pH. From left to right: M: Marker; pH: 7.0, 7.5 and 8.0. B. immunoblots of sAC protein expression and β-actin from Calu-3 cells exposed to HCO3--free culture medium at pH 7.0, 7.5 or 8.0. C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to 10 μM adenosine or 10 μM forskolin in the absence of bicarbonate. From left to right: M: Marker; SBF: standard bicarbonate free media; Ado: adenosine; FSK: forskolin. D: summary of the ratios of band densities (sAC/GAPDH) from A and C. Error bar indicates ± SE (n = 3).
Mentions: Changes in extracellular pH can regulate gene expression in renal cells in vitro [22]. Since culture medium pH varies with different [HCO3-], the up-regulation of sAC expression could be induced by changes in intracellular pH (pHi) secondary to differences in bath pH rather than HCO3-. The bath pH at different [HCO3-] was determined empirically. The bath pH ranges from 6.8 at 0 HCO3- to 7.8 at 80 HCO3-, if the culture medium contains 25 mM Hepes. A full pH unit difference in bath pH causes ~0.45 pH unit change in pHi [18]. In figure 3A, bath pH ranging from 7.0 to 8.0 did not change the expression of sAC mRNA in BCECs cultured in the absence of HCO3-. The ratios of competitive PCR product band densities (sAC/GAPDH) are summarized in Figure 3D. To examine sAC protein expression in different bath pH, sAC immunoblots were employed using Calu-3 cells. The effect of varying culture medium pH on sAC protein expression in Calu-3 cells is shown in figure 3B. From pH 7.0 to 8.0, equal density of sAC immunoblot bands were observed, which is consistent with the results shown in Fig. 3A.

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