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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 osmolarity and additional [NaCl] on sAC expression. RT-competitive PCR was performed using specific sAC primers and GAPDH internal standard primers from cultured BCECs. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different osmolarity (sucrose added). From left to right: M: Marker; osmolarity: 263, 272, 290, 321, 359, 383 (mmol/kg). B: summary of the ratios of band densities (sAC/GAPDH) from A. Error bar indicates ± SE (n = 3). C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different [NaCl]. From left to right: M: Marker; [NaCl]: 10, 20, 40, 60, 80 (mM). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).
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Figure 4: The effects of bath osmolarity and additional [NaCl] on sAC expression. RT-competitive PCR was performed using specific sAC primers and GAPDH internal standard primers from cultured BCECs. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different osmolarity (sucrose added). From left to right: M: Marker; osmolarity: 263, 272, 290, 321, 359, 383 (mmol/kg). B: summary of the ratios of band densities (sAC/GAPDH) from A. Error bar indicates ± SE (n = 3). C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different [NaCl]. From left to right: M: Marker; [NaCl]: 10, 20, 40, 60, 80 (mM). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).

Mentions: Since the osmolarity is increased with increasing NaHCO3 concentration, it is possible that regulation of sAC expression is caused by changing bath osmolarity rather than HCO3-. To exclude this possibility, we tested the effect of osmolarity alone (sucrose addition) or equimolar NaCl addition on the sAC expression. As shown in figure 4A,4B,4C,4D, the sAC expression was unaffected by increases of solution osmolarity alone or increased [NaCl], consistent with upregulation of sAC expression by 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 effects of bath osmolarity and additional [NaCl] on sAC expression. RT-competitive PCR was performed using specific sAC primers and GAPDH internal standard primers from cultured BCECs. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different osmolarity (sucrose added). From left to right: M: Marker; osmolarity: 263, 272, 290, 321, 359, 383 (mmol/kg). B: summary of the ratios of band densities (sAC/GAPDH) from A. Error bar indicates ± SE (n = 3). C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different [NaCl]. From left to right: M: Marker; [NaCl]: 10, 20, 40, 60, 80 (mM). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC411047&req=5

Figure 4: The effects of bath osmolarity and additional [NaCl] on sAC expression. RT-competitive PCR was performed using specific sAC primers and GAPDH internal standard primers from cultured BCECs. A: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different osmolarity (sucrose added). From left to right: M: Marker; osmolarity: 263, 272, 290, 321, 359, 383 (mmol/kg). B: summary of the ratios of band densities (sAC/GAPDH) from A. Error bar indicates ± SE (n = 3). C: RT-competitive PCR of sAC mRNA from cultured BCECs exposed to different [NaCl]. From left to right: M: Marker; [NaCl]: 10, 20, 40, 60, 80 (mM). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).
Mentions: Since the osmolarity is increased with increasing NaHCO3 concentration, it is possible that regulation of sAC expression is caused by changing bath osmolarity rather than HCO3-. To exclude this possibility, we tested the effect of osmolarity alone (sucrose addition) or equimolar NaCl addition on the sAC expression. As shown in figure 4A,4B,4C,4D, the sAC expression was unaffected by increases of solution osmolarity alone or increased [NaCl], consistent with upregulation of sAC expression by 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