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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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HCO3--induced up-regulation of sAC mRNA 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 [HCO3-]. From left to right: M: Marker; NC: negative control (without cDNA); [HCO3-]: 0, 5, 10, 20, 40, 60 and 80 (mM). 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 40 mM HCO3- with different incubation time. From left to right: M: Marker; NC: negative control (without cDNA); Time: 0, 3, 6, 12, 24, 48 (hours). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).
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Figure 1: HCO3--induced up-regulation of sAC mRNA 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 [HCO3-]. From left to right: M: Marker; NC: negative control (without cDNA); [HCO3-]: 0, 5, 10, 20, 40, 60 and 80 (mM). 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 40 mM HCO3- with different incubation time. From left to right: M: Marker; NC: negative control (without cDNA); Time: 0, 3, 6, 12, 24, 48 (hours). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).

Mentions: Our previous studies have shown that sAC is expressed in BCEC and distributed throughout the cytoplasm [18]. Bicarbonate directly activates sAC to increase [cAMP]i, which increases the phosphorylation of CFTR via cAMP-dependent PKA [18]. In the current study, we tested whether HCO3- can regulate the expression of sAC. RT-competitive PCR was performed to test the effect of HCO3- on sAC mRNA expression using specific sAC primers and internal standard GAPDH primers. First, we tested the regulation between [HCO3-] and sAC mRNA expression. As shown in figure 1A, sAC mRNA expression in BCECs increases with increasing [HCO3-] up to 40 mM. The decrease in the density of the GAPDH band is indicative of the competitive PCR format. The ratios of band densities (sAC/GAPDH) are summarized in figure 1B. Since 40 mM HCO3- maximally increased sAC mRNA expression, we chose this concentration to test sAC mRNA expression with different incubation times. As shown in figure 1C, sAC mRNA expression is significantly increased from 0 to 12 hour incubation time with no further increase at 24 or 48 hours. Theses results are summarized in Figure 1D.


[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)

HCO3--induced up-regulation of sAC mRNA 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 [HCO3-]. From left to right: M: Marker; NC: negative control (without cDNA); [HCO3-]: 0, 5, 10, 20, 40, 60 and 80 (mM). 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 40 mM HCO3- with different incubation time. From left to right: M: Marker; NC: negative control (without cDNA); Time: 0, 3, 6, 12, 24, 48 (hours). 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

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Figure 1: HCO3--induced up-regulation of sAC mRNA 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 [HCO3-]. From left to right: M: Marker; NC: negative control (without cDNA); [HCO3-]: 0, 5, 10, 20, 40, 60 and 80 (mM). 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 40 mM HCO3- with different incubation time. From left to right: M: Marker; NC: negative control (without cDNA); Time: 0, 3, 6, 12, 24, 48 (hours). D: summary of the ratios of band densities (sAC/GAPDH) from C. Error bar indicates ± SE (n = 3).
Mentions: Our previous studies have shown that sAC is expressed in BCEC and distributed throughout the cytoplasm [18]. Bicarbonate directly activates sAC to increase [cAMP]i, which increases the phosphorylation of CFTR via cAMP-dependent PKA [18]. In the current study, we tested whether HCO3- can regulate the expression of sAC. RT-competitive PCR was performed to test the effect of HCO3- on sAC mRNA expression using specific sAC primers and internal standard GAPDH primers. First, we tested the regulation between [HCO3-] and sAC mRNA expression. As shown in figure 1A, sAC mRNA expression in BCECs increases with increasing [HCO3-] up to 40 mM. The decrease in the density of the GAPDH band is indicative of the competitive PCR format. The ratios of band densities (sAC/GAPDH) are summarized in figure 1B. Since 40 mM HCO3- maximally increased sAC mRNA expression, we chose this concentration to test sAC mRNA expression with different incubation times. As shown in figure 1C, sAC mRNA expression is significantly increased from 0 to 12 hour incubation time with no further increase at 24 or 48 hours. Theses results are summarized in Figure 1D.

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