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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 protein expression. Cells were starved for HCO3- in HCO3--free DMEM for 48 hours at 37°C, followed by 24-hour incubation in DMEM with different [HCO3-] at 37°C. Cell lysates (60 μg/lane) were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with mouse anti-human monoclonal sAC primary antibody, then rinsed and reprobed using mouse anti-human β-actin antibody. A: blots of sAC protein expression and β-actin from BCECs exposed to 0, 10 or 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. B: blots of sAC protein expression and β-actin from Calu-3 cells exposed to 0 and 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. Error bar indicates ± SE. *different from 0 HCO3- (n = 3, p < 0.05).
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Figure 2: HCO3--induced up-regulation of sAC protein expression. Cells were starved for HCO3- in HCO3--free DMEM for 48 hours at 37°C, followed by 24-hour incubation in DMEM with different [HCO3-] at 37°C. Cell lysates (60 μg/lane) were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with mouse anti-human monoclonal sAC primary antibody, then rinsed and reprobed using mouse anti-human β-actin antibody. A: blots of sAC protein expression and β-actin from BCECs exposed to 0, 10 or 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. B: blots of sAC protein expression and β-actin from Calu-3 cells exposed to 0 and 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. Error bar indicates ± SE. *different from 0 HCO3- (n = 3, p < 0.05).

Mentions: To test whether up-regulated sAC mRNA can induce an increase in sAC protein expression, immunoblots were performed using a specific mouse anti-human sAC monoclonal antibody [9]. Both BCECs and calu-3 cells were tested. Calu-3 cells are a mixed phenotype of human bronchial epithelial and glandular cells often used to study cAMP dependent CFTR function [20,21]. RT-PCR demonstrated that sAC mRNA is also expressed in Calu-3 cells (data not shown). Figure 2 shows western blots using this antibody. For both cell types, bands at the expected 50 kD were found; some minor bands were seen with the cultured BCECs, but only a single band was seen from the calu-3 cells. sAC protein expression (figure 2A) in BCECs was increased by 45% and 87% in 10 and 40 mM HCO3- relative to 0 HCO3-, respectively. As shown in figure 2B, sAC protein expression was increased by 93% in 40 mM HCO3- relative to 0 HCO3- in Calu-3 cells, which is consistent with the results in BCECs.


[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 protein expression. Cells were starved for HCO3- in HCO3--free DMEM for 48 hours at 37°C, followed by 24-hour incubation in DMEM with different [HCO3-] at 37°C. Cell lysates (60 μg/lane) were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with mouse anti-human monoclonal sAC primary antibody, then rinsed and reprobed using mouse anti-human β-actin antibody. A: blots of sAC protein expression and β-actin from BCECs exposed to 0, 10 or 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. B: blots of sAC protein expression and β-actin from Calu-3 cells exposed to 0 and 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. Error bar indicates ± SE. *different from 0 HCO3- (n = 3, p < 0.05).
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Related In: Results  -  Collection

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Figure 2: HCO3--induced up-regulation of sAC protein expression. Cells were starved for HCO3- in HCO3--free DMEM for 48 hours at 37°C, followed by 24-hour incubation in DMEM with different [HCO3-] at 37°C. Cell lysates (60 μg/lane) were separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with mouse anti-human monoclonal sAC primary antibody, then rinsed and reprobed using mouse anti-human β-actin antibody. A: blots of sAC protein expression and β-actin from BCECs exposed to 0, 10 or 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. B: blots of sAC protein expression and β-actin from Calu-3 cells exposed to 0 and 40 mM HCO3-. Band density of sAC expression shown in the bar graph is relative to 0 HCO3-. Error bar indicates ± SE. *different from 0 HCO3- (n = 3, p < 0.05).
Mentions: To test whether up-regulated sAC mRNA can induce an increase in sAC protein expression, immunoblots were performed using a specific mouse anti-human sAC monoclonal antibody [9]. Both BCECs and calu-3 cells were tested. Calu-3 cells are a mixed phenotype of human bronchial epithelial and glandular cells often used to study cAMP dependent CFTR function [20,21]. RT-PCR demonstrated that sAC mRNA is also expressed in Calu-3 cells (data not shown). Figure 2 shows western blots using this antibody. For both cell types, bands at the expected 50 kD were found; some minor bands were seen with the cultured BCECs, but only a single band was seen from the calu-3 cells. sAC protein expression (figure 2A) in BCECs was increased by 45% and 87% in 10 and 40 mM HCO3- relative to 0 HCO3-, respectively. As shown in figure 2B, sAC protein expression was increased by 93% in 40 mM HCO3- relative to 0 HCO3- in Calu-3 cells, which is consistent with the results in BCECs.

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